Pituitary adenylate cyclase-activating polypeptide (PACAP) is an upstream regulator of prodynorphin mRNA expression in neurons

Neuroscience Letters 484 (2010) 174–177

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Pituitary adenylate cyclase-activating polypeptide (PACAP) is an upstream regulator of prodynorphin mRNA expression in neurons Ying Xu Dong a,c,1 , Mamoru Fukuchi a,1 , Minami Inoue a , Ichiro Takasaki b , Akiko Tabuchi a , Chun Fu Wu c , Masaaki Tsuda a,∗ a b c

Department of Biological Chemistry, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Sugitani 2630, Toyama 930-0194, Japan Life Science Research Center, University of Toyama, Sugitani 2630, Toyama 930-0194, Japan Department of Pharmacology, Shenyang Pharmaceutical University, 110016 Shenyang, China

a r t i c l e

i n f o

Article history: Received 8 July 2010 Received in revised form 11 August 2010 Accepted 16 August 2010 Keywords: PACAP Prodynorphin Neuropeptide PAC1 Gene expression Cortical neuron

a b s t r a c t Although dynorphins are widely involved in the control of not only nociceptive neurotransmission but also a variety of brain functions such as memory and emotion, no natural regulator for inducing the mRNA expression of prodynorphin (Pdyn), a precursor protein of dynorphins, is known. Using primary cultures of rat cortical neurons, we found that pituitary adenylate cyclase-activating polypeptide (PACAP), a member of the vasoactive intestinal polypeptide (VIP)/secretin/glucagon neuropeptide family, markedly induces Pdyn mRNA expression. PACAP was much more effective than VIP, indicating a major role for PAC1 in the PACAP-induced Pdyn mRNA expression. The increase in Pdyn mRNA expression was independent of de novo protein synthesis. Administration of forskolin, an activator for adenylate cyclase/protein kinase A (PKA), but not TPA, an activator for protein kinase C (PKC), induced Pdyn mRNA expression, suggesting a major role for PKA. The involvement of PKA was supported by the inhibition of PACAP-induced Pdyn mRNA expression upon addition of H89, an inhibitor for PKA. The PACAP-induced potentiation of NMDAR was involved in the mRNA expression of Bdnf or c-fos but not Pdyn. These results suggest PACAP to be an upstream regulator for inducing Pdyn mRNA expression through PKA. © 2010 Elsevier Ireland Ltd. All rights reserved.

Prodynorphin (Pdyn) is a precursor protein that generates endogenous opioid neuropeptides, including dynorphin A (Dyn A), dynorphin B (Dyn B), and big dynorphin (Big Dyn), consisting of Dyn A and Dyn B sequences [26]. Although dynorphins interact with -opioid receptors and exert analgesic effects [7], their prolonged administration induces hyperalgesia through N-methyld-aspartate receptors (NMDA-R) [13,28]. The expression of Pdyn mRNA and its protein products was found to be up-regulated in the spinal cord in animal models of inflammatory and neuropathic pain [9,18,24,31,32]. However, a natural regulator for inducing Pdyn mRNA is still unclear. On the other hand, chronic nociceptive responses are markedly reduced in mice lacking PAC1, a specific receptor for pituitary adenylate cyclase-activating polypeptide (PACAP) [11,25]. PACAP has been isolated from ovine hypothalamus extracts on the basis of its ability to stimulate the formation of cAMP in rat pituitary cells [19] and is widely expressed in the central nervous system [29], being involved in the control of pleiotropic

∗ Corresponding author at: Department of Biological Chemistry, Faculty of Pharmaceutical Sciences, University of Toyama, Sugitani 2630, Toyama 930-0194, Japan. Tel.: +81 76 434 7535; fax: +81 76 434 5048. E-mail address: [email protected] (M. Tsuda). 1 These authors contributed equally to this work. 0304-3940/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2010.08.044

physiological functions such as pain, synaptic plasticity, and emotional control. In the dorsal root ganglion (DRG), PACAP is expressed in small to medium-sized neurons projecting to superficial layers of the dorsal horn of the spinal cord, where PAC1 is expressed, and up-regulated after nerve transection [10]. Considering that the expression of PACAP is increased in DRG and that of dynorphins in the spinal dorsal horn, it seems possible that PACAP is a candidate regulator for stimulating Pdyn mRNA expression. In order to know the relationship between Pdyn and PACAP, in this study, we therefore investigated whether PACAP induces the Pdyn mRNA expression in neurons or not. Primary cultures of rat cortical neurons were prepared from the cerebral cortex of Sprague–Dawley rats as described previously [6]. The cerebral cortex was isolated from the brain at embryonic day 17. The cells were grown in Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen) containing 10% fetal calf serum (FCS), 100 U/mL of penicillin and 100 ␮g/mL of streptomycin. After 3 days, the medium was replaced with DMEM not containing FCS. Each experiment was performed after 5 days in culture. The proportion of neurons expressing PAC1 was estimated at about 60% by immunostaining the cortical neurons with anti-PAC1 antibody. Most of the PAC1-positive cells were positive for MAP2 (data not shown). Cortical neurons were stimulated with 100 nM PACAP, cultured for specified periods, and collected to prepare total RNA

Y.X. Dong et al. / Neuroscience Letters 484 (2010) 174–177

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Fig. 1. Effect of PACAP on Pdyn mRNA expression in cultured rat cortical neurons. A: (a) At 5 days in culture, cortical neurons were treated with 100 nM PACAP, and total RNA was extracted at the indicated times (h). The quantity of Pdyn mRNA was measured using quantitative RT-PCR. Mean ± S.E. n = 3–4. *p < 0.05 and **p < 0.01 versus control at the same time. (b) At 5 days in culture, neurons were treated with different concentrations of PACAP (black) or VIP (gray) for 3 h, and total RNA was extracted. The quantity of Pdyn mRNA was measured by quantitative RT-PCR. Mean ± S.E. n = 3. *p < 0.05 and **p < 0.01 versus control. B: A PAC1 antagonist, PACAP(6–38) (5 ␮M), or a VPAC1/VPAC2 antagonist, [Ac-Tyr1, d-Phe2] GRF (1–29) amide (AcGRF(1–29)a, 5 ␮M), was added to cultured neurons 10 min before the treatment with 100 nM PACAP. Total RNA was extracted 3 h after the treatment with PACAP, and the quantity of Pdyn mRNA was measured by quantitative RT-PCR. (a) Data was shown as a percent of control (in the absence of PACAP and antagonists). Mean ± S.E. n = 3–4. *p < 0.05 and **p < 0.01 versus control. ## p < 0.01 versus vehicle-treated sample with PACAP. (b) Data was shown as a fold-induction (compared to the sample without PACAP but with each antagonist or vehicle). Mean ± S.E. n = 3–4. C: Ten minutes before the treatment with PACAP, cells were pretreated with cycloheximide (CHX, 10 ␮g/mL). After the treatment with PACAP for 3 h, total RNA was extracted, and the quantity of Pdyn mRNA was measured by quantitative RT-PCR. Mean ± S.E. n = 3. *p < 0.05 and **p < 0.01 versus control. ## p < 0.01 versus vehicle.

for measuring the expression of Pdyn mRNA. Quantitative RTPCR was performed using Mx3000p (Stratagene), according to methods described previously [6]. For an internal control, Gapdh cDNA was amplified, and the levels of each transcript were normalized to Gapdh. Primer sequences for measuring mRNA were: for Pdyn, 5 -TTGTGTTCCCTGTGTGCAGT-3 and 5 -AGTGCCCA GTAGCTCAGATT-3 ; for c-fos, 5 -GTTTCAACGCGGACTACGAG-3 and 5 -AGCGTATCTGTCAGCTCCCT-3 ; for Bdnf, 5 -CCACCAGGTGA GAAGAGTGATGACC-3 and 5 -GCCCATTCACGCTCTCCA-3 ; and for Gapdh, 5 -ATCGTGGAAGGGCTCATGAC-3 and 5 -TAGCCCAGG ATGCCCTTTAGT-3 . All values represent the mean ± S.E. of results from a number of separate experiments performed in duplicate, as indicated in the corresponding figures. Statistical analyses were performed using Student’s t-test followed by the F-test with the probability level for significance set to p < 0.05. To investigate whether PACAP induces the expression of Pdyn mRNA in neurons, we examined the changes in Pdyn mRNA levels in primary cultures of rat cortical neurons stimulated with PACAP, using the quantitative RT-PCR method. As shown in Fig. 1A(a), the mRNA expression increased 1 h after the treatment of cultured neurons with 100 nM PACAP, peaked at 3 h, and then decreased 6 h later, indicating that the expression of Pdyn mRNA is transiently induced by a direct effect of PACAP. We next investigated the responses of Pdyn mRNA expression to the administration of PACAP or VIP and compared them. As shown in Fig. 1A(b), the mRNA level began to increase at 100 pM PACAP, peaking at 10 nM PACAP, whereas it took 100 nM for VIP to have any effect, the difference corresponding to that in the affinity for PAC1 [29]. These results indicate that PACAP induces Pdyn mRNA expres-

sion by acting on PAC1 in culture, though, at higher concentrations of PACAP, VPAC1/VPAC2 could also be involved. PACAP receptors are classified into two types based on affinity for PACAP and VIP [4,29]. PAC1, a type I receptor, exhibits high affinity for PACAP, and much lower affinity for VIP. VPAC1 and VPAC2, type II receptors, possess similar affinity for PACAP and VIP. To determine which receptors are involved in the increase in Pdyn mRNA expression induced by PACAP at 100 nM, we added PACAP(6–38) and [Ac-Tyr1, d-Phe2] GRF(1–29) amide (AcGRF(1–29)a), antagonists for PAC1 and VPAC1/VPAC2, respectively, in excess amounts to the medium (final concentration; 5 ␮M). Although the basal level of Pdyn mRNA expression slightly increased upon the treatment of cultured cells with PACAP(6–38) or AcGRF(1–29)a (Fig. 1B(a)), PACAP-induced Pdyn mRNA expression was partially repressed by pretreatment with these antagonists (Fig. 1B(b); Fold-induction; Vehicle, 7.41 ± 0.856; PACAP(6–38), 4.62 ± 0.559; and AcGRF(1–29)a, 4.61 ± 0.254). The antagonistic effect of PACAP(6–38) on the PACAP-induced Pdyn mRNA expression seemed to be relatively low. Inhibitory effect of PACAP(6–38) on the PACAP-induced c-fos and Bdnf mRNA expression were also observed (Supplementary Fig. 1). It has been suggested that PACAP(6–38) acts as a VPAC2 agonist besides its antagonistic effect on PAC1 [29]. The low antagonistic effect of PACAP(6–38) on the PACAP-induced Pdyn mRNA expression might be due to such a side-effect of PACAP(6–38). In the rat Pdyn promoter, the binding site for activator protein-1 (AP-1), an inducible transcription factor, is found [20]. Therefore, to investigate whether or not the PACAP-induced Pdyn mRNA expression is dependent on de novo protein synthesis of such an inducible transcription factor, we examined the effect of cyclohex-

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Fig. 2. Effect of protein kinase inhibitors or a blockade of NMDA-R on PACAP-induced Pdyn mRNA expression. A: (a) Cultured rat cortical cells were treated with forskolin (10 ␮M), TPA (500 ng/mL), forskolin plus TPA, or 100 nM PACAP for 3 h, and total RNA was extracted for measuring the quantity of Pdyn mRNA. Mean ± S.E. n = 4–5. **p < 0.01 versus control. (b) A PKA inhibitor, H89 (10 ␮M), was added to cultured neurons 10 min before the treatment with 100 nM PACAP. Total RNA was extracted 3 h after the treatment with PACAP. Mean ± S.E. n = 4. ***p < 0.001 versus control. ### p < 0.001 versus vehicle-treated sample with PACAP. B: An antagonist for NMDA-R, APV, was added to cultured neurons 10 min before the treatment with 100 nM PACAP. Total RNA was extracted 3 h after the treatment with PACAP, and the quantities of Pdyn (a), c-fos (b) and Bdnf (c) mRNA were measured by quantitative RT-PCR. Mean ± S.E. n = 3–4. **p < 0.01 and ***p < 0.001 versus control. # p < 0.05 and ## p < 0.01 versus vehicle-treated sample with PACAP.

imide (CHX), an inhibitor of protein synthesis. As shown in Fig. 1C, CHX (10 ␮g/mL) actually enhanced the expression, a phenomenon known as “super-induction” [23], indicating that the Pdyn mRNA expression induced by PACAP is not dependent on de novo protein synthesis. Next we focused on the intracellular signaling pathways involved in the PACAP-induced Pdyn mRNA expression. PACAP mainly activates adenylate cyclase (AC) and phospholipase C (PLC), resulting in an increase in second messengers, cAMP, IP3 , and calcium [4,29]. Therefore, we particularly focused on the signaling pathways including protein kinase A (PKA) or protein kinase C (PKC). Stimulation of cortical neurons with 10 ␮M forskolin, an activator for AC, to the cultures of cortical neurons enhanced the Pdyn mRNA expression while treatment with 500 ng/mL of TPA, a potent activator for PKC, did not (Fig. 2A(a)). The increase in Pdyn mRNA expression induced by PACAP was completely inhibited by pretreatment with 10 ␮M H89, an inhibitor of PKA (Fig. 2A(b)). However, further addition of TPA with forskolin tended to enhance the expression compared to levels obtained with forskolin alone (Fig. 2A(a)), the mechanism for which is unclear. In any case, it is evident that the PACAP-induced Pdyn mRNA expression is mediated by the activation of the PKA pathway. It has already been demonstrated that the formation of cAMP in cortical cells was dose-dependently stimulated by PACAP, peaking at 10 nM, but by VIP at higher concentrations than 100 nM [16], corresponding to the dose–responses of Pdyn mRNA induction (Fig. 1B(b)). Taken together, it is possible that the cAMP/PKA pathway is a main one to control the PACAP-induced Pdyn mRNA expression. A single intrathecal injection of Dyn A induces allodynia through the NMDA-R [13,28]. In addition, evidence has recently emerged that stimulation of PAC1 with PACAP enhances the

responses of NMDA-R through signaling pathways including not only G␣s/AC/PKA [30] but also G␣q/PLC/PKC [15], leading to the expression of immediate–early genes such as Bdnf and c-fos, which is repressed by a blockade of NMDA-R [16,22]. Thus, the activation of PAC1 indirectly modulates the activity of NMDA-R. To determine whether the activation of NMDA-R is necessary for the PACAPinduced Bdnf, c-fos, or Pdyn mRNA expression, we used a potent antagonist for NMDA-R, APV. The PACAP-induced Bdnf and c-fos mRNA expression was inhibited by 200 ␮M APV (Fig. 2B(b) and (c)), whereas the expression of Pdyn mRNA induced by PACAP was not affected in the presence of APV (Fig. 2B(a)), indicating no involvement of NMDA-R activation in the PACAP-induced Pdyn mRNA expression. Four cAMP-response elements (CREs) were found in the Pdyn promoter [5]. Using VP16-CREB mice, in which a constitutively active CREB is expressed in the forebrain, Pdyn expression was found to be up-regulated in transgenic mice [1], indicating that Pdyn is a target of CREB. Binding sites for functional AP-1 and other factors were also found in the rat Pdyn promoter region [5,17,20]. On the other hand, downstream regulatory element antagonist modulator (DREAM), a calcium-regulated transcriptional repressor, is involved in the suppression of Pdyn expression. DREAM-knockout mice had elevated levels of Pdyn mRNA and dynorphin protein, and displayed markedly reduced responses in several pain models [3]. Considering that the PACAP-induced increase in Pdyn mRNA expression is controlled by the PKA pathway (Fig. 2A), it seems possible that CREB is responsible for the increase. However, further investigation is needed to clarify how not only CREB but also other transcription factors are involved in the control of the PACAP-induced Pdyn mRNA expression.

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Given that PACAP is involved in the control of pain-related behavior [11,14] and Pdyn expression in the spinal cord was upregulated by inflammation and nociception [9,24,31,32], it seems plausible for the PACAP-induced Pdyn gene expression to be involved in nociceptive neurotransmission. Moreover, dynorphins have regulatory roles in numerous functional pathways of the brain. In line with their broad distribution in the hippocampus, amygdala, hypothalamus and striatum, dynorphins are thought to be involved in learning and memory, emotional control, and stress responses [26]. In practice, it has already been demonstrated that dynorphins are involved in memory acquisition, and anxiety-like and locomotor behavior in mice [12], and defects of dynorphins or -opioid receptors are pathophysiologically related to epilepsy, addiction, depression and schizophrenia [2,26,27]. Moreover, a disruption of PACAP expression in the brain has already been suggested to be associated with a cause for psychiatric disorders including schizophrenia [8,21]. Therefore, it is highly possible that a cascade of PACAP-induced gene expression, including the expression of Pdyn, could be involved in regulating not only nociception but also a variety of neuronal functions and diseases in the central nervous system. Acknowledgements We thank Dr. A. Sasaki (Department of Applied Pharmacology, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Japan) for helpful discussions. This study was supported in part by a Grant-in-aid for Scientific Research from the Ministry of Education, Science, Sports and Culture, Japan (Project Number 20390023, M.T.), and the Mitsubishi Foundation (M.T.). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.neulet.2010.08.044. References [1] A. Barco, S. Patterson, J.M. Alarcon, P. Gromova, M. Mata-Roig, A. Morozov, E.R. Kandel, Gene expression profiling of facilitated L-LTP in VP16-CREB mice reveals that BDNF is critical for the maintenance of LTP and its synaptic capture, Neuron 48 (2005) 123–137. [2] W.A. Carlezon Jr., C. Béguin, J.A. DiNieri, M.H. Baumann, M.R. Richards, M.S. Todtenkopf, R.B. Rothman, Z. Ma, D.Y. Lee, B.M. Cohen, Depressive-like effects of the kappa-opioid receptor agonist salvinorin A on behavior and neurochemistry in rats, J. Pharmacol. Exp. Ther. 316 (2006) 440–447. [3] H.Y. Cheng, G.M. Pitcher, S.R. Laviolette, I.Q. Whishaw, K.I. Tong, L.K. Kockeritz, T. Wada, N.A. Joza, M. Crackower, J. Goncalves, I. Sarosi, J.R. Woodgett, A.J. Oliveira-dos-Santos, M. Ikura, D. van der Kooy, M.W. Salter, J.M. Penninger, DREAM is a critical transcriptional repressor for pain modulation, Cell 108 (2002) 31–43. [4] L. Dicksona, K. Finlaysonb, VPAC and PAC receptors: from ligands to function, Pharmacol. Ther. 121 (2009) 294–316. [5] J. Douglass, A.A. McKinzie, K.M. Pollock, Identification of multiple DNA elements regulating basal and protein kinase A-induced transcriptional expression of the rat prodynorphin gene, Mol. Endocrinol. 8 (1994) 333–344. [6] M. Fukuchi, A. Tabuchi, M. Tsuda, Activity-dependent transcriptional activation and mRNA stabilization for cumulative expression of pituitary adenylate cyclase-activating polypeptide mRNA controlled by calcium and cAMP signals in neurons, J. Biol. Chem. 279 (2004) 47856–47865. [7] J.S. Han, C.W. Xie, Dynorphin: potent analgesic effect in spinal cord of the rat, Sci. Sin. B. 27 (1984) 169–177. [8] R. Hashimoto, H. Hashimoto, N. Shintani, S. Chiba, S. Hattori, T. Okada, M. Nakajima, K. Tanaka, N. Kawagishi, K. Nemoto, T. Mori, T. Ohnishi, H. Noguchi, H. Hori, T. Suzuki, N. Iwata, N. Ozaki, T. Nakabayashi, O. Saitoh, A. Kosuga, M. Tatsumi, K. Kamijima, D.R. Weinberger, H. Kunugi, A. Baba, Pituitary adenylate cyclaseactivating polypeptide is associated with schizophrenia, Mol. Psychiatry 12 (2007) 1026–1032.

177

[9] M.J. Iadarola, L.S. Brady, G. Draisci, R. Dubner, Enhancement of dynorphin gene expression in spinal cord following experimental inflammation: stimulus specificity, behavioral parameters and opioid receptor binding, Pain 35 (1988) 313–326. [10] H. Jongsma, N. Danielson, F. Sundler, M. Kanje, Alteration of PACAP distribution and PACAP receptor binding in the rat sensory nervous system following sciatic nerve transection, Brain Res. 853 (2000) 186–196. [11] H. Jongsma, L.M. Pettersson, Y. Zhang, M.K. Reimer, M. Kanje, A. Waldenström, F. Sundler, N. Danielsen, Markedly reduced chronic nociceptive response in mice lacking the PAC1 receptor, Neuroreport 12 (2001) 2215–2219. [12] A. Kuzmin, N. Madjid, L. Terenius, S.O. Ogren, G. Bakalkin, Big dynorphin, a prodynorphin-derived peptide produces NMDA receptor-mediated effects on memory, anxiolytic-like and locomotor behavior in mice, Neuropsychopharmacology 31 (2006) 1928–1937. [13] T.M. Laughlin, T.W. Vanderah, J. Lashbrook, M.L. Nichols, M. Ossipov, F. Porreca, G.L. Wilcox, Spinally administered dynorphin A produces long-lasting allodynia: involvement of NMDA but not opioid receptors, Pain 72 (1997) 253–260. [14] T. Mabuchi, N. Shintani, S. Matsumura, E. Okuda-Ashitaka, H. Hashimoto, T. Muratani, T. Minami, A. Baba, S. Ito, Pituitary adenylate cyclase-activating polypeptide is required for the development of spinal sensitization and induction of neuropathic pain, J. Neurosci. 24 (2004) 7283–7291. [15] D.S. Macdonald, M. Weerapura, M.A. Beazely, L. Martin, W. Czerwinski, J.C. Roder, B.A. Orser, J.F. MacDonald, Modulation of NMDA receptors by pituitary adenylate cyclase activating peptide in CA1 neurons requires G␣q, protein kinase C, and activation of Src, J. Neurosci. 25 (2005) 11374–11384. [16] J.L. Martin, D. Gasser, P.J. Magistretti, Vasoactive intestinal peptide and pituitary adenylate cyclase-activating polypeptide potentiate c-fos expression induced by glutamate in cultured cortical neurons, J. Neurochem. 65 (1995) 1–9. [17] C.T. McMurray, K.M. Pollock, J. Douglass, Cellular and molecular analysis of opioid peptide gene expression, NIDA Res. Monogr. 126 (1992) 113–131. [18] I. Merchenthaler, J.L. Maderdrut, P. Cianchetta, P. Shughrue, D. Bronstein, In situ hybridization histochemical localization of prodynorphin messenger RNA in the central nervous system of the rat, J. Comp. Neurol. 384 (1997) 211–232. [19] A. Miyata, A. Arimura, R.R. Dahl, N. Minamino, A. Uehara, L. Jiang, M.D. Culler, D.H. Coy, Isolation of a novel 38 residue-hypothalamic polypeptide which stimulates adenylate cyclase in pituitary cells, Biochem. Biophys. Res. Commun. 64 (1989) 567–574. [20] J.R. Naranjo, B. Mellstrom, M. Achaval, P. Sassone-Corsi, Molecular pathways of pain: Fos/Jun-mediated activation of a noncanonical AP-1 site in the prodynorphin gene, Neuron 6 (1991) 607–617. [21] A. Nicot, T. Otto, P. Brabet, E.M. DiCicco-Bloom, Altered social behavior in pituitary adenylate cyclase-activating polypeptide type I receptor-deficient mice, J. Neurosci. 24 (2004) 8786–8795. [22] G. Pellegri, P.J. Magistretti, J.L. Martin, VIP and PACAP potentiate the action of glutamate on BDNF expression in mouse cortical neurones, Eur. J. Neurosci. 10 (1998) 272–280. [23] J. Radulovic, N.C. Tronson, Protein synthesis inhibitors, gene superinduction and memory: too little or too much protein? Neurobiol. Learn. Mem. 89 (2008) 212–218. [24] M.A. Ruda, M.J. Iadarola, L.V. Cohen, W.S. Young 3rd., In situ hybridization histochemistry and immunocytochemistry reveal an increase in spinal dynorphin biosynthesis in a rat model of peripheral inflammation and hyperalgesia, Proc. Natl. Acad. Sci. U. S. A. 85 (1988) 622–626. [25] K. Sándor, K. Bölcskei, J.J. McDougall, N. Schuelert, D. Reglodi, K. Elekes, G. Petho, E. Pintér, J. Szolcsányi, Z. Helyes, Divergent peripheral effects of pituitary adenylate cyclase-activating polypeptide-38 on nociception in rats and mice, Pain 141 (2009) 143–150. [26] C. Schwarzer, 30 Years of dynorphins-new insights on their functions in neuropsychiatric diseases, Pharmacol. Ther. 123 (2009) 353–370. [27] Y. Shirayama, H. Ishida, M. Iwata, G.I. Hazama, R. Kawahara, R.S. Duman, Stress increases dynorphin immunoreactivity in limbic brain regions and dynorphin antagonism produces antidepressant-like effects, J. Neurochem. 90 (2004) 1258–1268. [28] T.W. Vanderah, T. Laughlin, J.M. Lashbrook, M.L. Nichols, G.L. Wilcox, M.H. Ossipov, T.P. Malan Jr., F. Porreca, Single intrathecal injections of dynorphin A or des-Tyr-dynorphins produce long-lasting allodynia in rats: blockade by MK-801 but not naloxone, Pain 68 (1996) 275–281. [29] D. Vaudry, A. Falluel-Morel, S. Bourgault, M. Basille, D. Burel, O. Wurtz, A. Fournier, B.K. Chow, H. Hashimoto, L. Galas, H. Vaudry, Pituitary adenylate cyclase-activating polypeptide and its receptors: 20 years after the discovery, Pharmacol. Rev. 61 (2009) 283–357. [30] R. Yaka, D.Y. He, K. Phamluong, D. Ron, Pituitary adenylate cyclase-activating polypeptide (PACAP (1–38)) enhances N-methyl-d-aspartate receptor function and brain-derived neurotrophic factor expression via RACK1, J. Biol. Chem. 278 (2003) 9630–9638. [31] R.X. Zhang, L. Lao, J.T. Qiao, M.A. Ruda, Effects of aging on hyperalgesia and spinal dynorphin expression in rats with peripheral inflammation, Brain Res. 999 (2004) 135–141. [32] R.X. Zhang, B. Liu, L. Lao, J.T. Qiao, M.A. Ruda, Spinal preprodynorphin mRNA expression in neonatal rats following peripheral inflammation, Brain Res. 1038 (2005) 238–242.