G Model

ARTICLE IN PRESS

NSL 30555 1–5

Neuroscience Letters xxx (2014) xxx–xxx

Contents lists available at ScienceDirect

Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet

Somatostatin 4 receptor activation modulates TPRV1 currents in dorsal root ganglion neurons

1

2

3

Q1

4

Louise Gorham ∗ , Stefan Just, Henri Doods Boehringer Ingelheim Pharma GmbH & Co. KG, Dept. CNS Diseases Research Germany, Birkendorfer Strasse 65, 88397 Biberach an der Riss, Germany

5

6 7 8 9 10 11 12

h i g h l i g h t s • • • • •

Influence of a SSTR4 agonist, J-2156, on TRPV1 activity in DRG neurons was assessed. J-2145 concentrationally inhibited capsaicin induced calcium influx. J-2156 concentrationally inhibited capsaicin induced sodium influx. J-2145 inhibition of capsaicin induced calcium influx is augmented after CFA treatment. This inhibition identifies a contributing mechanism to the analgesic effects of J-2156.

13

14 28

a r t i c l e

i n f o

a b s t r a c t

15 16 17 18 19 20

Article history: Received 24 March 2014 Received in revised form 16 April 2014 Accepted 28 April 2014 Available online xxx

21

27

Keywords: sst4 Receptor Somatostatin agonist J-2145 DRG TRPV1 GPCR

29Q2

1. Introduction

22 23 24 25 26

30 31 32 33 34 35 36

Somatostatin (sst) is a cyclic neuropeptide known to have inhibitory roles in the central nervous system. It exerts its biological effects via the activation of the 5 sst receptor subtypes, which belong to the family of G-protein coupled receptors (GPCR). This peptide has analgesic properties, specifically via the activation of the sst4 receptor subtype. Although this is established, the precise molecular mechanisms causing this have not yet been fully elucidated. This research aimed to identify a possible anti-nociceptive mechanism, showing functional links to the transient receptor potential vanilloid type 1 (TRPV1) within the pain processing pathway. Calcium imaging and whole cell voltage clamp experiments were conducted on DRG neurons prepared from adult rats, utilizing capsaicin stimulations and the sst4 receptor specific agonist J2156. The complete Freund’s adjuvant (CFA) inflammatory pain model was used to examine if effects are augmented in pain conditions. The sst4 receptor agonist J-2156 was able significantly to inhibit capsaicin induced calcium and sodium influx, where the effect was more potent after CFA treatment. This inhibition identifies a contributory molecular mechanism to the analgesic properties of sst4 receptor activation. © 2014 Elsevier Ireland Ltd. All rights reserved.

The neuropeptide somatostatin (sst) is widely distributed throughout the body, having critical roles within the central nervous system (CNS) [22]. It exerts such a broad range of biological effects by associating with the 5 receptor subtypes, sst1–5 , which all belong to the family of G-protein coupled receptors (GPCRs) [17]. This neuropeptide has recently been implicated in analgesia, where activation of sst receptors results in inhibition of nociceptive

Abbreviations: ATP, adenosine triphosphate; CFA, complete Freund’s adjuvant; CNS, central nervous system; DMEM, Dulbecco’s modified Eagle medium; DRG, dorsal root ganglion neurons; GPCR, G protein coupled receptor; HEPES, hydroxyethyl piperazineethanesulfonic; PKA, protein kinase A; sst, somatostatin; TRPV1, transient receptor potential vanilloid 1. ∗ Corresponding author. Tel.: +49 7351 54 7028; fax: +49 7351 54 5128. E-mail address: louise catherine [email protected] (L. Gorham).

behaviors [11]. Similarly sst analogs inhibit neuronal activity [20] and decrease responses of joint mechanoreceptors to noxious rotation [24]. Of the five sst receptor subtypes it is the sst4 receptor that is thought to be responsible for the analgesic properties associated with sst. This was concluded on the basis of pharmacological data [21], as well as knock out animal studies [10]. The sst4 receptor is expressed in the peripheral pain regulatory pathways, specifically DRG neurons [2]. Nociception involves the transmission of nerve impulses along primary afferent neurons, known as dorsal root ganglion neurons (DRGs). These cells contain a diverse group of ligand-gated and voltage-gated ion channels which are able to transduce noxious stimulation into depolarizations, allowing nociceptive signals to be conducted along neuronal axons [13]. Although it is well established that activation of sst4 receptors results in analgesia, the precise molecular mechanisms involved have yet to be fully elucidated. A family of channels associated with pain onset is the transient receptor potential channels, for

http://dx.doi.org/10.1016/j.neulet.2014.04.042 0304-3940/© 2014 Elsevier Ireland Ltd. All rights reserved.

Please cite this article in press as: L. Gorham, et al., Somatostatin 4 receptor activation modulates TPRV1 currents in dorsal root ganglion neurons, Neurosci. Lett. (2014), http://dx.doi.org/10.1016/j.neulet.2014.04.042

37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54

G Model NSL 30555 1–5

ARTICLE IN PRESS L. Gorham et al. / Neuroscience Letters xxx (2014) xxx–xxx

2

77

example, the transient receptor potential vanilloid type 1 (TRPV1). These channels are predominantly expressed in nociceptive sensory neurons [5], where they have essential roles in the detection and modulation of painful stimuli [7]. TRPV1 channels are known to be activated by many different stimuli including mechanical, chemical, thermal and pH changes [5]. Additionally they can be modulated by GPCRs. Activation of Gi proteins results in the inhibition of adenylate cyclase, leading to inhibition of cyclic adenosine monophosphate (cAMP) formation, causing a reduced activation of protein kinase A (PKA) [14]. The lack of PKA activation ultimately prevents TRPV1 sensitization [12]. This mechanism is reversed by pertussis toxin, where the inhibitory effect on cAMP via sst4 receptor activation is abolished following pretreatment with the toxin [9]. The objective of this study was to investigate the actions of sst4 receptor activation on TRPV1 channel signaling. In the present study we show that sst4 receptor activation modulates TRPV1 channel currents in DRG neurons, which was abolished following pertussis toxin pre-treatment. As the sst4 receptor agonist J-2156 is particularly potent in the complete Freund’s adjuvant (CFA) model of inflammatory pain [21], we investigated if this functional link was augmented in this pain model. We found the effects were more potent post CFA injection.

78

2. Methods

79

2.1. Animals and CFA-induced inflammation

55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76

91

Adult male Crl:WI (Han) rats (Charles River, Germany) weighing 300–350 g were used in all experiments. Inflammation was induced by a 50 ␮l subcutaneous injection of 25 ␮g Complete Freund’s Adjuvant (CFA, Sigma, USA) into the plantar surface of the left hind paw. For sham experiments 50 ␮l of buffer was injected. All intraplantar injections were performed under brief isoflurane (Abbott GmbH, Germany) anesthesia. All animal protocols were authorized by the Local Animal Care and Use Committee and carried out according to the local animal care guidelines, AAALAC regulations, and the USDA Animal Welfare Act. These followed the guidelines provided by Committee for Research and Ethical Issues of IASP. DRG neurons were sampled 24 h after CFA injection.

92

2.2. Cell culture: DRG neurons

80 81 82 83 84 85 86 87 88 89 90

106

Adult DRG neuron cultures were prepared from 6 to 8 week old Crl:WI (Han) (Charles River, Germany) male rats. From CFA injected rats only L4-L6 DRGs from the inflamed side were removed and cultured. DRGs were removed and placed into DMEM (C.C. Pro., Germany, FM-13-L). Digestion of cells was achieved using 4 mg/ml collagenase (Gibco, UK, 17104-019) and 2 mg/ml papain (Sigma, Germany, P4762) at 37 ◦ C for 75 min. This reaction was terminated by aspiration, and addition of 10% FCS (Gibco, UK, 10500-064). The cells were dissociated by mechanical agitation and plated onto 0.1 mg/ml poly-l-lysine (Sigma, Germany, P-7886) and 2 ␮g/ml laminin (Sigma, Germany, C2020-IMG) coated cover slips (Thermo Scientific, Germany). The cells were stored at 37 ◦ C, 10% carbon dioxide and with a humidity of 95%. Recordings were made within 24 h of plating.

107

2.3. Calcium imaging

93 94 95 96 97 98 99 100 101 102 103 104 105

108 109 110 111 112 113

The DRG neurons were pre-loaded with the Ca2+ -sensitive fluorophore, Fluo-4-AM, 2 ␮M (Invitrogen, CA) and 1 mM probenecid (Sigma, Germany) for one hour prior to imaging. The cells were then washed in Ringer buffer (140 mM NaCl; 5 mM KCl; 10 mM glucose; 10 mM HEPES; 1.8 mM CaCl2 ; 0.8 mM MgCl2 ; pH 7.4 using NaOH) until use. The cells were used within one hour of pre-loading. Cells

were imaged through a plane neofluar 20×/0,5 objective, using an Axiovert 200M microscope with inbuilt camera (Zeiss). Excitation was achieved at 488 nm with an argon laser, and images were collected, at an emission of 510 nm, through a long pass filter, using the computer software Laser Scanning Microscope 510 META version 3,2 SP2 (Zeiss). The fluorescent signal was optimized by the detector gain and amplifier offset. The cells were mounted on a custom made perfusion chamber. Activation of TRPV1 channels was achieved via application of 30 nM capsaicin for 30 s. 2.4. Electrophysiology Transmembrane currents were recorded by whole-cell voltageclamp at −60 mV holding potential, using an EPC 10 amplifier, with TIDA 5.2 software (HEKA Electronics, Germany) and a PCI-1600 interface (HEKA Electronics, Germany). Data were low-pass filtered at 2.9 kHz and sampled at 20 kHz. Patch pipettes were pulled from borosilicate glass capillaries (1.5 mm outer diameter) on a Sutter Instruments P-97 puller (Sutter Instruments, USA), and had a resistance of 3–5 M. The pipette solution consisted of: 145 mM KCl; 10 mM glucose; 10 mM HEPES; 1 mM MgCl2 , with the addition of 2 mM Na2 ATP and 0.2 mM NaGTP on the day of testing. The cells were bathed in calcium free Ringer’s solution containing: 140 mM NaCl; 5 mM KCl; 10 mM glucose; 10 mM HEPES; 1 mM MgCl2 . The pH was adjusted to 7.4 using NaOH and the osmolarity remained between 300 and 320 mOsmol/l. Activation of the TRPV1 channels was achieved with a 300 nM capsaicin application for 5 s, where cells were considered responsive if the inward current magnitude was at least 100 pA. The pipette potential was zeroed before seal formation; care was taken to maintain membrane access resistance as low as possible, between 3 and 7 M. Command voltage protocols and data acquisition were performed by TIDA 5.2 (HEKA Electronics, Germany). 2.5. Statistics Statistical analysis was carried out using prism5 software. Data are expressed as mean ± S.E.M. Normal distribution was testing using the Kolmogorov–Smirnov test. One-way ANOVA measurements, followed by Bonferroni post-hoc tests, allowed for significance to be determined. The logIC50 was calculated to determine the concentration of compound which produces 50% of the maximal inhibition. Raw data traces are shown by normalization of the fluorescent intensity [F/F0 ]. 3. Results In order to determine if the sst4 receptor is functionally coupled to TRPV1 channels in DRG neurons we used two experimental techniques; calcium imaging and patch clamp. A previous described sst4 receptor specific agonist J-2156 [9] was utilized, where the TRPV1 channels were activated using capsaicin. Initially the concentration of capsaicin needed for stimulation of DRG neurons was established. The cells were exposed to capsaicin for 30 s; both 30 nM and 100 nM were able to induce a calcium influx, where no significant difference is calculated (data not shown). We therefore chose to continue with 30 nM capsaicin for future experiments, as with this concentration less desensitization was observed. With calcium imaging experiments 2 protocols were run. The first was a double stimulation protocol, where the cells were exposed twice to 30 nM capsaicin for 30 s, and the ratio between the two peaks determined. Application of capsaicin resulted in an increase in intracellular calcium levels. A control experiment was run, where the second stimulation had a slightly reduced

Please cite this article in press as: L. Gorham, et al., Somatostatin 4 receptor activation modulates TPRV1 currents in dorsal root ganglion neurons, Neurosci. Lett. (2014), http://dx.doi.org/10.1016/j.neulet.2014.04.042

114 115 116 117 118 119 120 121 122

123

124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144

145

146 147 148 149 150 151 152 153

154

155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172

G Model NSL 30555 1–5

ARTICLE IN PRESS L. Gorham et al. / Neuroscience Letters xxx (2014) xxx–xxx

3

Fig. 1. J-2156 inhibits capsaicin induced calcium currents in rat DRG neurons. (A) Statistical analysis (ANOVA) of the effects of J-2156. The sst4 receptor specific agonist significantly inhibited TRPV1 current at 10 nM, 100 nM and 1000 nM (P < 0.01, P < 0.0001, P < 0.0001 vs. control, respectively). Capsazepine at 10 ␮M significantly inhibited the TRPV1 current (P < 0.0001 vs. control). Averages of the response to 30 nM capsaicin are shown (B). These results are represented as the ratio of the two stimulations, S1:S2. N = 24–53. 173 174 175 176 177 178 179 180 181 182 183 184 185 186 187 188 189 190 191 192 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 217

evoked calcium transient (Fig. 1). When the sst4 receptor agonist was applied for 3 min prior to the second stimulation, the calcium peak was reduced. J-2156 caused a significant decrease in the capsaicin induced calcium transient at 10 nM (P < 0.01 vs. control), 100 nM and 1000 nM (P < 0.0001 vs. control). A decrease of 27 ± 5%, 45 ± 6% and 45 ± 5% was seen, respectively (n = 23–53 cells; Fig. 1A). No effect was seen at 1 nM J-2156 application. The effect of capsazepine, a competitive antagonist of TRPV1 channels [3] was also determined. This caused a significant decrease in capsaicin evoked calcium transient at 10 ␮M (P < 0.0001 vs. control; n = 24–53; Fig. 1A) of 61 ± 8%. The raw recordings are shown in Fig. 1B; although there was not complete recovery, the slope showing recovery is steeper post compounds application in comparison to the control experiment. So this inhibitory effect is noticeable almost directly after compound application by the heightened rate of recovery seen. The second protocol involved pre-incubation of the cells for 5 min with the compound. The cells were then exposed to 30 nM capsaicin for 30 s and 3 min later depolarization was induced by 80 mM extracellular potassium solution. The percentage of the potassium response was calculated. The control cells resulted in a calcium influx by both stimulations, where there was only a slight increase in the potassium response (Fig. 2A). Both the sst4 receptor agonist, J-2156 and the TRPV1 antagonist, capsazepine, reduced the capsaicin evoked calcium influx, however, they still fully responded to the potassium. J-2156 caused a significant decrease in the capsaicin induced calcium transient at 10 nM (P < 0.01 vs. control), 100 nM and 1000 nM (P < 0.001 vs. control), a decrease of 42 ± 9%, 62 ± 6% and 64 ± 9% was seen, respectively (n = 17–56 cells; Fig. 2B). The logIC50 is −8.269. At a concentration of 10 ␮M, capsazepine caused a significantly reduced calcium influx compared to the control (P < 0.001 vs. control; n = 16–25; Fig. 2B), where reduction of 92 ± 2% was seen. To confirm modulation is via the PKA pathway, experiments were run utilizing pertussis toxin, an agent known to uncouple Gi proteins from GPCRs. J-2156 was tested at 100 nM, as this concentration had maximal efficacy. The cells were pretreated with 100 ng/ml pertussis toxin and the second protocol run. The inhibition of capsaicin induced calcium influx was abolished following pretreatment with the toxin (P = 0.3209 vs. control; n = 56; Fig. 2B). This second protocol was used to establish if the sst4 receptor induced TRPV1 inhibition is augmented after CFA treatment. J2156 caused a significant decrease in the capsaicin induced calcium transient at concentrations as low as 0.1 nM (P < 0.01 vs. control; n = 55; Fig. 2C). Significant effects were seen at 1 nM (P < 0.01 vs.

control), 10 nM, 100 nM and 1000 nM (P < 0.001 vs. control), a decrease of 40 ± 5%, 52 ± 6%, 57 ± 4% and 55 ± 6% was seen, respectively (n = 50–65 cells; Fig. 2C). The logIC50 is −10.79. However sham treated animals reacted in the same way as naïve, where no inhibition was seen at 0.1 nM and maximal effect at 100 nM of 60 ± 6% (P < 0.001 vs. control; n = 41; Fig. 2C). With the electrophysiology experiments a higher concentration of capsaicin was used. It was necessary to use 300 nM capsaicin as when 30 nM was used the number of cells that responded was low. Those cells which responded had an induced sodium current of 85 ± 16 pA at 30 nM, compared to 384 ± 116 pA with 300 nM (data not shown). This current was considered too small for an appropriate assay window. A protocol was run where 300 nM capsaicin was applied for 5 s at two time points; 60 and 360 s. The last application was run in the presence of either vehicle control or the compound and the ratio of the two peaks was calculated. Application of capsaicin resulted in an induced sodium current. A control experiment was run, where the final stimulation had a slightly reduced effect (Fig. 3). When the SSTR4 agonist was applied for 5 min prior to the final peak the capsaicin response was further reduced. J-2156 at 100 nM induced a significant decrease in TRPV1 activity (P < 0.01 vs. control) of 25 ± 6% (n = 9–15; Fig. 3A). Capsazepine at 10 ␮M also induced a significant decrease in TRPV1 activity (P < 0.0001 vs. control) of 67 ± 7% (n = 9–15; Fig. 3A). 4. Discussion In the present study we were able to show that activation of the sst4 receptor results in inhibition of TRPV1 channels within DRG neurons. Two experimental techniques were conducted utilizing the sst4 receptor agonist J-2156 [9], and the TRPV1 agonist capsaicin [3]. These results showed that the J-2156 evokes a concentration dependent decrease in TRPV1 cation current in DRG neurons. The sst4 receptor agonist, J-2156, is a particularly potent analgesia for inflammatory pain [21]. We therefore tested whether the observed inhibition of TRPV1 current by sst4 receptor activation was augmented after CFA treatment. These results showed that the potency of J-2156 is higher during inflammatory pain compared with naïve animals, although the overall efficacy is similar. TPRV channels play key roles within nociception. Relating directly to pain transmission, activation of these channels allows the movement of sodium and calcium ions into the cell and potassium ions out of the cell, resulting in an inward depolarizing current. This would lead to cell excitation, triggering action potential propagation and transmission of pain signals [7]. Relating to

Please cite this article in press as: L. Gorham, et al., Somatostatin 4 receptor activation modulates TPRV1 currents in dorsal root ganglion neurons, Neurosci. Lett. (2014), http://dx.doi.org/10.1016/j.neulet.2014.04.042

218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 233 234 235 236 237 238 239 240 241

242

243 244 245 246 247 248 249 250 251 252 253 254 255 256 257 258 259 260

G Model NSL 30555 1–5 4

ARTICLE IN PRESS L. Gorham et al. / Neuroscience Letters xxx (2014) xxx–xxx

Fig. 3. J-2156 inhibits capsaicin induced sodium currents in rat DRG neurons. (A) Statistical analysis (ANOVA) of the effects of the sst4 receptor specific agonist J-2156 100 nM and Capsazepine 10 ␮M, both compounds significantly inhibited the TRPV1 current (P < 0.01, P < 0.0001 vs. control, respectively). (B) Examples of whole-cell current to 300 nM capsaicin stimulations: control (top), J-2156 (middle) and capsazepine (bottom). These results are represented as the ratio of the two stimulations, S1:S2. N = 9–15.

Fig. 2. J-2156 inhibition of TRPV1 currents is augmented after CFA treatment. (A) Protocol description and average response for naïve recordings. (B) Statistical analysis (ANOVA) of the effects of J-2156 on naïve DRG neurons. The sst4 receptor specific agonist significantly inhibited TRPV1 current at 10 nM, 100 nM and 1000 nM (P < 0.001, P < 0.0001 vs. control, respectively). IC50 = −8.269 M. Capsazepine at 10 ␮M significantly inhibited the TRPV1 current (P < 0.0001 vs. control). Preincubation with 100 ng/ml pertussis toxin abolished the J-2156 TRPV1 inhibition. (C) Statistical analysis (ANOVA) of the effects of J-2156 on CFA treated DRG neurons, where significant inhibition of TRPV1 current was seen at concentrations as low as 0.1 nM (P < 0.05 vs. control); maximal effects were reached by 10 nM (P < 0.0001 vs. control). IC50 = −10.79 M. Sham treated DRGs responded in the same way as naïve DRGs. These results are represented as the % of potassium response. N = 25–56.

261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277

expression, TRPV1 channels are predominantly expressed in nociceptive specific neurons [5], these are sensitized and up-regulated during tissue damage, confirming roles within pain [25]. Supporting this Caterina et al. [4] reported that TRPV1 knock-out mice showed no response to vanilloid induced pain, a reduced response to acidification and a greatly reduced response to noxious heat. These data therefore identify that inhibition of TRPV1 channels provides a useful mechanism for analgesia. Furthermore TRPV1 has been implicated in inflammatory pain. This channel is sensitized by inflammatory agents [6] and is essential for thermal hyperalgesia in inflammation but not neuropathy [7]. Furthermore expression of this channel is increased in all types of DRG neurons after CFA injection [8], giving an explanation regarding the augmented effect we see in inflamed DRG neurons. Inhibition of TRPV1 channels has been implicated in the mechanism underlying the anti-nociception produced by wellestablished compounds. This is not only true for other GPCRs

including opioid receptors [8]; but also capsazepine a competitive antagonist of the TRPV1 channel [3]. Capsazepine is able to inhibit nocifensive and hyperalgesic responses to capsaicin and other inflammatory agents by direct binding to the channel, therefore offering analgesia via blockage of TRPV1 mediated signals [19]. Our results confirm the findings of Pintér et al. [18], who conducted in-vivo experiments using the sst4 receptor specific agonist TT-232. This compound was able significantly to reduce both nociceptive and inflammatory responses evoked by capsaicin, indicating that sst4 receptor agonists affect TRPV1 activity, where our results confirm direct modulation in an ex-vivo model. In the calcium imaging experiments two protocols were run, seen in Figs. 1B and 2A, one a double stimulation protocol and the other a pre-exposure protocol. The effects of capsazepine are more pronounced in the pre-exposure protocol (Fig. 2A); in this case the cells have no previous exposure to capsaicin, meaning capsazepine does not have to compete for binding. This enhanced effect of the second protocol is also true for the sst4 receptor agonist J2156. This compound does not directly bind to the TRPV1 channel, where no binding is seen at concentrations of up to 10 ␮M (unpublished data). However another explanation could be the calcium induced calcium release effect [23] impacting the double stimulation protocol. The remaining calcium present in the cell from the first stimulation contributes to the second, thus reducing the effect of the compounds; whereas in the second protocol the compound effect is shown directly by the first peak. With the electrophysiology experiments it was necessary to use a 100 fold higher concentration of capsaicin to get a relevant response. The two experimental techniques are measuring different ions. In the calcium imaging experiments the response can be enhanced by release of ions from intracellular stores [23]. Similarly, the effect of capsaicin may be different for the two ions due to the pore size of the channels [1]. It is assumed that many TRP channels, upon activation, are subject to pore dilation, meaning the pore size is changed. Pore dilation results in an increased selectivity of calcium ions and an increased calcium current through the channel [16], again indicating how a lower capsaicin concentration causes a larger current in the calcium imaging experiments.

Please cite this article in press as: L. Gorham, et al., Somatostatin 4 receptor activation modulates TPRV1 currents in dorsal root ganglion neurons, Neurosci. Lett. (2014), http://dx.doi.org/10.1016/j.neulet.2014.04.042

278 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297 298 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315

G Model NSL 30555 1–5

ARTICLE IN PRESS L. Gorham et al. / Neuroscience Letters xxx (2014) xxx–xxx

321

In conclusion, our results indicate that activation of sst4 receptor in DRG neurons leads to inhibition of transient receptor potential vanilloid type 1 (TRPV1) channels, where the effect is augmented after CFA treatment. These observations demonstrate a contributory mechanism underlying the analgesic effects of peripherally acting sst4 receptor agonist J-2156 in inflammatory pain.

Q3 322

Uncited reference

316 317 318 319 320

323

324

325 326 327

328

329 330 331 332 333 334 335 336 337 338 339 340 341 342 343 344 345 346 347 348 349 350 351 352 353 354

[15]. Acknowledgments The authors would like to thank Luke Bryden, Rosmarie Ewen, Carsten Hecker, Benjamin Jaehnke, Gert Kramer and Margot Weiland for their technical assistance. References [1] T.G. Banke, S.R. Chaplan, A.D. Wickenden, Dynamic changes in the TRPA1 selectivity filter lead to progressive but reversible pore dilation, Am. J. Physiol.: Cell Physiol. 298 (2010) C1457–C1468. [2] K.J. Bär, U. Schurigt, A. Scholze, G. Segond Von Banchet, N. Stopfel, R. Bräuer, K.J. Halbhuber, H.G. Schaible, The expression and localization of somatostatin receptors in dorsal root ganglion neurons of normal and monoarthritic rats, Neuroscience 127 (2004) 197–206. [3] S. Bevan, S. Hothi, G. Hughes, I.F. James, H.P. Rang, K. Shah, C.S. Walpole, J.C. Yeats, Capsazepine: a competitive antagonist of the sensory neurone excitant capsaicin, Br. J. Pharmacol. 107 (1992) 544–552. [4] M.J. Caterina, A. Leffler, A.B. Malmberg, W.J. Martin, J. Trafton, K.R. PetersenZeitz, M. Koltzenburg, A.I. Basbaum, D. Julius, Impaired nociception and pain sensation in mice lacking the capsaicin receptor, Science 288 (2000) 306–313. [5] M.J. Caterina, M.A. Schumacher, M. Tominaga, T.A. Rosen, J.D. Levine, D. Julius, The capsaicin receptor: a heat-activated ion channel in the pain pathway, Nature 389 (1997) 816–824. [6] D.N. Cortright, A. Szallasi, Biochemical pharmacology of the vanilloid receptor TRPV1. An update, Eur. J. Biochem. 271 (2004) 1814–1819. [7] J.B. Davis, J. Gray, M.J. Gunthorpe, J.P. Hatcher, P.T. Davey, P. Overend, M.H. Harries, J. Latcham, C. Clapham, K. Atkinson, S.A. Hughes, K. Rance, E. Grau, A.J. Harper, P.L. Pugh, D.C. Rogers, S. Bingham, A. Randall, S.A. Sheardown, Vanilloid receptor-1 is essential for inflammatory thermal hyperalgesia, Nature 405 (2000) 183–187. [8] J. Endres-Becker, P.A. Heppenstall, S.A. Mousa, D. Labuz, A. Oksche, M. Schäfer, C. Stein, C. Zöllner, Mu-opioid receptor activation modulates transient receptor potential vanilloid 1 (TRPV1) currents in sensory neurons in a model of inflammatory pain, Mol. Pharmacol. 71 (2007) 12–18.

5

[9] M. Engstrom, J. Tomperi, K. El-Darwish, M. Ahman, J.M. Savola, S. Wurster, Superagonism at the human somatostatin receptor subtype 4, J. Pharmacol. Exp. Ther. 312 (2005) 332–338. [10] Z. Helyes, E. Pinter, K. Sandor, K. Elekes, A. Banvolgyi, D. Keszthelyi, E. Szoke, D.M. Toth, Z. Sandor, L. Kereskai, G. Pozsgai, J.P. Allen, P.C. Emson, A. Markovics, J. Szolcsanyi, Impaired defense mechanism against inflammation, hyperalgesia, and airway hyperreactivity in somatostatin 4 receptor gene-deleted mice, Proc. Nat. Acad. Sci. U.S.A. 106 (2009) 13088–13093. [11] Z. Helyes, M. Than, G. Oroszi, E. Pinter, J. Nemeth, G. Keri, J. Szolcsanyi, Anti-nociceptive effect induced by somatostatin released from sensory nerve terminals and by synthetic somatostatin analogues in the rat, Neurosci. Lett. 278 (2000) 185–188. [12] P.Y. Law, Y.H. Wong, H.H. Loh, Molecular mechanisms and regulation of opioid receptor signaling, Annu. Rev. Pharmacol. Toxicol. 40 (2000) 389–430. [13] E.W. McCleskey, M.S. Gold, Ion channels of nociception, Annu. Rev. Physiol. 61 (1999). [14] D.P. Mohapatra, C. Nau, Desensitization of capsaicin-activated currents in the vanilloid receptor TRPV1 is decreased by the cyclic AMP-dependent protein kinase pathway, J. Biol. Chem. 278 (2003) 50080–50090. [15] I. Nagy, P. Sántha, G. Jancsó, L. Urbán, The role of the vanilloid (capsaicin) receptor (TRPV1) in physiology and pathology, Eur. J. Pharmacol. 500 (2004) 351–369. [16] B. Nilius, J. Prenen, G. Owsianik, Irritating channels: the case of TRPA1, J. Physiol. 589 (2011) 1543–1549. [17] Y.C. Patel, M. Greenwood, R. Panetta, N. Hukovic, S. Grigorakis, L.A. Robertson, C.B. Srikant, Molecular biology of somatostatin receptor subtypes, Metabolism 45 (1996) 31–38. [18] E. Pintér, Z. Helyes, J. Nemeth, R. Porszasz, G. Petho, M. Than, G. Keri, A. Horvath, B. Jakab, J. Szolcsanyi, Pharmacological characterisation of the somatostatin analogue TT-232: effects on neurogenic and non-neurogenic inflammation and neuropathic hyperalgesia, Naunyn Schmiedebergs Arch. Pharmacol. 366 (2002) 142–150. [19] H.K. Rami, M. Thompson, P. Wyman, J.C. Jerman, J. Egerton, S. Brough, A.J. Stevens, A.D. Randall, D. Smart, M.J. Gunthorpe, J.B. Davis, Discovery of small molecule antagonists of TRPV1, Bioorg. Med. Chem. Lett. 14 (2004) 3631–3634. [20] J. Sandkühler, Q.G. Fu, C. Helmchen, Spinal somatostatin superfusion in vivo affects activity of cat nociceptive dorsal horn neurons: comparison with spinal morphine, Neuroscience (England) 34 (1990) 565–576. [21] K. Sandor, K. Elekes, A. Szabo, E. Pinter, M. Engstrom, S. Wurster, J. Szolcsanyi, Z. Helyes, Analgesic effects of the somatostatin sst(4) receptor selective agonist J-2156 in acute and chronic pain models, Eur. J. Pharmacol. 539 (2006) 71–75. [22] S. Schulz, M. Handel, M. Schreff, H. Schmidt, V. Hollt, Localization of five somatostatin receptors in the rat central nervous system using subtype-specific antibodies, J. Physiol. (Paris) 94 (2000) 259–264. [23] A. Shmigol, A. Verkhratsky, G. Isenberg, Calcium-induced calcium release in rat sensory neurons, J. Physiol. 489 (Pt 3) (1995) 627–636. [24] F. Silveri, P. Morosini, D. Brecciaroli, C. Cervini, Intraarticular injection of somatostatin in knee osteoarthritis—clinical-results and IGF-1 serum levels, Int. J. Clin. Pharmacol. Res. 14 (1994) 79–85. [25] M. Tominaga, M.J. Caterina, A.B. Malmberg, T.A. Rosen, H. Gilbert, K. Skinner, B.E. Raumann, A.I. Basbaum, D. Julius, The cloned capsaicin receptor integrates multiple pain-producing stimuli, Neuron 21 (1998) 531–543.

Please cite this article in press as: L. Gorham, et al., Somatostatin 4 receptor activation modulates TPRV1 currents in dorsal root ganglion neurons, Neurosci. Lett. (2014), http://dx.doi.org/10.1016/j.neulet.2014.04.042

355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 372 373 374 375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 402 403 404 405 406