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Home , Big dynorphin, Prodynorphin

Professor Tomas Ekström and Docent Georgy Bakalkin 2/9/2009 ______

Final Report S-26:05

PHARMACOTHERAPY OF CHRONIC PAIN. A NOVEL APPROACH THAT TARGETS

THE UBIQUITIN-PROTEASOME SYSTEM.

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Table of Content Page ______

Description of main results 1

General conclusions 9

List of published and submitted papers 9 supported by the AFA grant ______

The main results of our studies supported by AFA Forsäkring for the time period from July 2003 to July 2007 may be put into three interrelated groups:

1. A critical role of in pain. Identification and characterization of the most active endogenous pathological agents inducing pain among fragments (Papers ## 1, 7). 2. (A) Development of novel decoy that scavenge A and prevent NMDA-receptor mediated effects (Paper # 9). (B) Inhibition of neuropathic pain by small molecules that target the ubiquitin proteasome system (UPS) and dynorphins secretion (Paper # 18). 3. Characterization of the prodynorphin system ant its functions in the human and animal brain (Papers ## 3, 5, 6, 8, 10, 14, 15). Cellular and molecular mechanisms of pronociceptive effects of dynorphins (Papers ## 4, 12, 19).

Section 1. A critical role of dynorphins in pain. Identification and characterization of the most active endogenous pathological agents inducing pain among prodynorphin fragments (Papers ## 1, 7).

The diversity of ligands for a particular receptor may provide a greater dynamic range of functional responses while maintaining selectivity in receptor activation. 1 Professor Tomas Ekström and Docent Georgy Bakalkin 2/9/2009 ______Dynorphin A (Dyn A), and (Dyn B) are endogenous peptides that activate the κ- (KOR). We characterized interactions of (Big Dyn), a 32 amino acid prodynorphin derived peptide consisting of Dyn A and Dyn B, with human (h) KOR, μ-(hMOR) and δ-(hDOR) opioid receptors and opioid receptor-like receptor 1 (hORL1) expressed in cells transfected with respective cDNA. Big Dyn and Dyn A demonstrated roughly similar affinity for binding to hKOR that was higher than that of Dyn B. Dyn A was more selective for hKOR over hMOR, hDOR and hORL1 than Big Dyn while Dyn B demonstrated low selectivity. In contrast, Big Dyn activated G proteins through KOR with much greater potency, efficacy and selectivity than other dynorphins. There was no correlation between the rank order of the potency for the KOR-mediated activation of G proteins and the binding affinity of dynorphins for KOR. The rank of the selectivity for the activation of G proteins through hKOR and of the binding to this receptor also differed. Immunoreactive Big Dyn was detected using the combination of RIA and HPLC in the human nucleus accumbens, caudate nucleus, hippocampus and cerebrospinal fluid with the ratio of Big Dyn and Dyn B being approximately 1 to 3. The presence in the brain implies that Big Dyn along with other dynorphins is processed from prodynorphin and secreted from neurons. Collectively, the high potency and efficacy and the relative abundance suggest that Big Dyn may play a role in the KOR-mediated activation of G proteins.

To identify the most active pronociceptive peptides we analyzed the ability of several prodynorphin fragments to induce nociceptive behavior after their intrathecal injection into mice. Big dynorphin, a 32 aa peptide consisting of dynorphins A and B, appeared to be the most potent peptide in this respect. It was 100-fold more patent than in inducing pain, whereas dynorphin B did not induce any changes. Big dynorphin-induced nociceptive behavior was mediated through the activation of the NMDA receptor ion- channel complex by acting on the polyamine recognition site, and does not involve opioid, non-NMDA glutamate receptor mechanisms, or tachykinin receptors. We further demonstrated that N-ethylmaleimide (NEM), a cysteine protease inhibitor induces nociceptive behavior through the inhibition of degradation of endogenous big dynorphin in mice. Intratechal administration of this compound produced a characteristic behavioral response, the biting and licking of the hindpaws and the tail, along with slight hindlimb scratching directed toward the flank. Behavior induced by NEM was inhibited by pretreatment with anti-dynorphin A or anti-dynorphin B antiserum. Both antisera also prevented nociceptive effects of big dynorphin. Importantly, NEM was not able to induce nociceptive behavior in prodynorphin knockout mice. The NEM-induced nociceptive behavior was abolished by ifenprodil, arcaine and agmatine, the antagonists of the polyamine recognition site on the NMDA receptor ion-channel complex, and by MK-801, an NMDA ion-channel blocker. In contrast, Ro25-6981, an antagonist of a NMDA receptor subtype (the NR2B subunit), had no effect. Collectively, our results demonstrate that endogenous prodynorphin-derived peptides are pronociceptive in uninjured animals.

Section 2. (A) Development of novel decoy peptides that scavenge dynorphin A and prevent NMDA-receptor mediated effects (Paper # 9). (B) Inhibition of neuropathic pain by small molecules that target the ubiquitin proteasome system (UPS) and dynorphins secretion (Paper # 18).

2 Professor Tomas Ekström and Docent Georgy Bakalkin 2/9/2009 ______

(A) Collaboration with Drs. Amina Woods and Toni Shippenberg). We pursued studies aimed to design small molecules that target dynorphin A, the main pathological agent in neuropathic pain (Paper # 9).

Prodynorphin-derived peptides elicit various pathological effects including neurological dysfunction and cell death. These actions are reduced by N-methyl-D-aspartate receptor (NMDAR) but not opioid receptor antagonists suggesting NMDAR-mediation. In this study, we show that a conserved epitope (KVNSEEEEEDA) of the NR1 subunit of the NMDAR binds dynorphin A noncovalently. Synthetic peptides containing this epitope form stable complexes with dynorphin A and prevent the potentiation of NMDAR-gated currents produced by dynorphin A. They attenuate dynorphin A-evoked cell death in spinal cord and prevent, as well as reverse, dynorphin A-induced paralysis and allodynia. The data reveal a novel mechanism whereby prodynorphin-derived peptides facilitate NMDAR function and produce neurotoxicity. Furthermore, they suggest that synthetic peptides that bind dynorphin A, thus preventing their interaction with NMDAR, may be novel therapeutic agents for the treatment of spinal cord injury.

(B) The core part of the project (Paper # 18) Specific aims of this section were (1) to evaluate the pharmacological potential of proteasome inhibitors in prevention and reversion of neuropathic pain, and (2) to establish the optimal dosing schedule and route of administration, and 3) to evaluate toxicity of these compounds (Collaboration with Prof. Frank Porreca).

The following main research goals have been reached: 1. We have confirmed and extended our previous observations that proteasome inhibitors of two chemical classes inhibit and prevent the development of neuropathic pain. We also generate data showing that proteasome inihibitors are not toxic at doses used. Particularly, we found that proteasome inhibitors (i) produced a time- and dose-related prevention of nerve injury– induced tactile and thermal hypersensitivity when administered concurrently with nerve injury; (ii) elicited a reversal of nerve injury–induced sensory thresholds when treatment was initiated after full manifestation of the neuropathic pain state; (iii) did not produce sensory or motor disturbances at doses that modify neuropathic pain; (iv) partially or completely normalized multiple neurochemical and morphological consequences of nerve injury in both the peripheral and central nervous systems.

We also demonstrate that 1) degradation of proteins by the proteasome is essential for both the initiation (prevention experiments) and maintenance (reversion experiments) of nerve injury-induced neuropathic pain; 2) neuropathic pain is abolished as long as the proteasome is inhibited (experiments with long-term exposure to proteasome inhibitors); and 3) effects on neuropathic pain are reversible whereas no signs of compensation or tolerance are developed upon sustained administration of proteasome inhibitors. We also observed that 1) inhibition of a small fraction of the proteasome is sufficient to prevent neuropathic pain; 2) proteasome inhibitors block the upregulation of two neurotransmitter systems (CGRP and dynorphins) both critical for spinal sensitization and neuropathic pain; and 3) the effects of proteasome inhibitors on neuropathic pain are mediated through multiple neurochemical systems, the status of which is normalized upon inhibition of the proteasome.

3 Professor Tomas Ekström and Docent Georgy Bakalkin 2/9/2009 ______

The facts that proteasome inhibitors do not change non-nociceptive reflexes, slightly inhibit nociceptive reflexes in sham-operated animals, do not produce motor dysfunction and do not induce cell death in the spinal cord suggest that these compounds are not neurotoxic under conditions used. This is consistent with previous studies demonstrating that administration of proteasome inhibitors into animals (i.p. at a 10 mg/kg/day dose, for 31 days) (Sun et al., 2001) and humans was relatively well tolerated (Kisselev and Goldberg, 2001; Orlowski et al., 2002; Richardson et al., 2003; Aghajanian et al., 2002).

Summary of the study: Chronic pain is maintained in part by long-lasting neuroplastic changes in synapses and several proteins critical for synaptic plasticity are degraded by the ubiquitin–proteasome system (UPS). Here, we show that proteasome inhibitors administered intrathecally or subcutaneously prevented the development and reversed nerve injury-induced pain behavior. They also blocked pathological pain induced by sustained administration of or spinal injection of dynorphin A, an endogenous mediator of chronic pain. Proteasome inhibitors blocked mechanical allodynia and thermal hyperalgesia in all three pain models although they did not modify responses to mechanical stimuli, but partially inhibited responses to thermal stimuli in control rats. In the spinal cord, these compounds abolished the enhanced capsaicin-evoked gene-related peptide (CGRP) release and dynorphin A upregulation, both elicited by nerve injury. Model experiments demonstrated that the inhibitors may act directly on dynorphin-producing cells, blocking dynorphin secretion. Thus, the effects of proteasome inhibitors on chronic pain were apparently mediated through several cellular mechanisms indispensable for chronic pain, including those of dynorphinArelease and postsynaptic actions, and ofCGRPsecretion. Levels of severalUPSproteins were reduced in animals with neuropathic pain, suggesting that UPS downregulation, like effects of proteasome inhibitors, counteracts the development of chronic pain. The inhibitors did not produce marked or disabling motor disturbances at doses that were used to modify chronic pain. These results suggest that the UPS is a critical intracellular regulator of pathological pain, and that UPS-mediated protein degradation is required for maintenance of chronic pain and nociceptive, but not non-nociceptive responses in normal animals.

Altogether, our results indicate that the UPS represents a novel target for efficient treatment of clinical neuropathic pain. The most selective availabe drugs that target the UPS may, however, be limited due to potential neurotoxicity after chronic i.t. administration in the spinal cord. Therefore, the design of less toxic inhibitors is essential.

Section 3. Characterization of the prodynorphin system ant its functions in the human and animal brain (Papers ## 3, 5, 6, 8, 10, 14, 15). Cellular and molecular mechanisms of pronociceptive effects of dynorphins (Papers ## 4, 12, 19).

To efficiently prevent pronociceptive effects of dynorphins the knowledge of cellular/molecular mechanisms of the non-opioid and opioid effects of dynorphins is required. Studies described in Papers ## 4 and 12 led to two novel complementary hypotheses of activation of nociceptive neurons by these peptides.

4 Professor Tomas Ekström and Docent Georgy Bakalkin 2/9/2009 ______The classical view postulates that precursors in neurons are processed into mature in the somatic trans-Golgi network (TGN) and in secretory vesicles during axonal transport. Here we show that prodynorphin (PDYN), precursor to dynorphin opioid peptides, is predominantly located in axon terminals and dendrites in hippocampal and striatal neurons. The molar content of unprocessed PDYN was much greater than that of dynorphin peptides in axon terminals of PDYN-containing neurons projecting to the CA3 region of the hippocampus and in the striatal projections to the ventral tegmental area. Electron microscopy showed coexistence of PDYN and dynorphins in the same axon terminals with occasional codistribution in individual dense core vesicles. Thus, the precursor protein is apparently stored at presynaptic sites. In comparison with the hippocampus and , PDYN and dynorphins were more equally distributed between neuronal somata and processes in the amygdala and cerebral cortex, suggesting regional differences in the regulation of trafficking and processing of the precursor protein. Potassium-induced depolarization activated PDYN processing and secretion of opioid peptides in neuronal cultures and in a model cell line. Regulation of PDYN storage and processing at synapses by neuronal activity or extracellular stimuli may provide a local mechanism for regulation of synaptic transmission.

Prodynorphin peptides generated on activation of D1 receptors (D1Rs) in the nucleus accumbens (Acb) are important contributors to both conditional place aversion and sensitization to the locomotor activating effects of psychostimulants. The location of prodynorphin, specifically as related to the dopaminergic (DA) inputs and D1Rs in the Acb is fundamental for establishing the physiologically relevant sites. To determine these sites, we examined the electron microscopic dual-labeling of prodynorphin and D1R or tyrosine hydroxylase (TH), a marker of catecholamine terminals in the rat Acb shell. This subregion is targeted by mesolimbic DA inputs affecting reward-aversion responses and locomotor activity. Prodynorphin was prominently localized to large (100-200 nm) granular aggregates in somatodendritic and axonal profiles using immunogold or immunoperoxidase labeling methods. In somata and dendrites, prodynorphin was often found in punctate clusters in the cytoplasm. Of the total prodynorphin-labeled dendrites, 62.9% (39/62) expressed D1Rs, which were largely located on dendritic plasma membranes. In comparison with dendrites, many more axon terminals were prodynorphin-labeled. Of the prodynorphin-immunoreactive terminals, 14.6% (67/459) contained D1R-labeling. Prodynorphin terminals formed symmetric synapses with D1R- labeled or unlabeled dendrites and also apposed TH-containing axon terminals. Our results provide ultrastructural evidence that in the Acb shell, D1Rs have subcellular distributions conductive to local dendritic or less commonly, axonal regulation of dynorphin production from the prodynorphin precursor. We also implicate axonal appositions and axodendritic synapses in the reciprocal regulation between dynorphin and D1R-targeted neurons in the Acb shell. These associations may substantially contribute to the dynorphin-mediated behavioral effects of dopamine.

How do dynorphins activate nociceptive neurons? Analysis of the primary structure demonstrated that dynorphins are similar to a group of peptides known as cell penetrating peptides and high in content of basic and hydrophobic amino acid residues. Cell penetrating peptides including penetratin and Tat can translocate across the plasma membrane into the cytoplasm and the cell nucleus. The similarity suggests that dynorphins are also capable of penetrating into cells, and this process may be relevant for

5 Professor Tomas Ekström and Docent Georgy Bakalkin 2/9/2009 ______interneuronal communication in the CNS. The capacity of dynorphins to translocate into cells was tested with confocal fluorescence microscopy/immunolabeling in live and fixed neuronal and non-neuronal cells. Translocation of big dynorphin and dynorphin A into the cytoplasm was characterized by substantial cytoplasmic labeling with minimal signal in the cell nucleus and on the plasma membrane. Translocated peptides were associated with the endoplasmic reticulum but not with the Golgi apparatus or clathrin-coated endocytic versicles. Consistent with these findings, rapid kinetics of fluorescent peptide entry into the cytoplasm was revealed by fluorescence correlation spectroscopy on live cells. Big dynorphin translocated into cells was largely intact as shown by mass- spectrometry. Dynorphin B and the central big dynorphin fragment, which retains all basic amino acid residues, did not enter the cells. Translocation was not mediated via opioid receptors. Fluorescence and circular dichroism spectroscopy showed that dynorphin A and big dynorphin to a larger extent than dynorphin B and the central big dynorphin fragment adopt α-helical structure upon interaction with charged phospholipid vesicles. The potential to form helical structure rather than the net positive charge correlates with the ability of dynorphin A and big dynorphin to translocate into cells. Translocation across the plasma membrane may represent an alternative mechanism by which dynorphins can signal information to the cell interior. Dynorphins penetration into cells may be based on their ability to make pores in the membrane. We tested this hypothesis in collaboration with Prof. Astrid Gräslund, Stockholm University. We observed that big dynorphin and dynorphin A, but not dynorphin B, cause leakage effects in large unilamellar phospholipid vesicles (LUVs). The effects parallel the previously studied potency of dynorphins to translocate through biological membranes. Calcein leakage caused by dynorphin A from LUVs with varying POPG/POPC molar ratios was promoted by higher phospholipid headgroup charges, suggesting that electrostatic interactions are important for the calcein leakage effects. A possibility that dynorphins generate non-opioid excitatory effects by inducing perturbations in the lipid bilayer of the plasmamembrane is discussed. The translocation potential and the ability to form pores also strongly correlate with the potential of dynorphin to induce abnormal pain in an animal model.

In the next investigation, we show that big dynorphin, dynorphin A and to some extent dynorphin A(1-13), but not dynorphin B, allow calcium to enter into large unilamellar phospholipid vesicles with partly negative headgroups. The effects parallel the previously studied potency of dynorphins to translocate through biological membranes and to cause calcein leakage from large unilamellar phospholipid vesicles. There is no calcium ion influx into vesicles with zwitterionic headgroups. We have also investigated if the dynorphins can translocate through the vesicle membranes and estimated the relative strength of interaction of the peptides with the vesicles by fluorescence resonance energy transfer. The results show that dynorphins do not translocate in this membrane model system however there is a strong electrostatic contribution to the interaction of the peptides with the membrane model system.

Thus, our current hypothesis is that dynorphins, through pore formation allowing Ca2+ ion influx, induce neuronal depolarization and activation of the NMDA receptors resulting in abnormal pain.

6 Professor Tomas Ekström and Docent Georgy Bakalkin 2/9/2009 ______Transcription from multiple promoters along with alternative mRNA splicing constitutes the basis for cell-specific gene expression and mRNA and protein diversity. The prodynorphin gene (PDYN) gives rise to prodynorphin (PDYN), precursor to dynorphin opioid peptides that regulate diverse physiological functions including pain. In this study, we characterized PDYN transcripts and proteins in the adult human brain and studied PDYN processing and intracellular localization in model cell lines. Nine PDYN mRNAs were identified in the human brain; two of the transcripts, FL1 and FL2, encode the full- length PDYN. The dominant, FL1 transcript shows high expression in limbic-related structures such as the nucleus accumbens and amygdala. The second, FL2 transcript is only expressed in few brain structures such as the claustrum and . FL- PDYN was identified for the first time in the brain as the dominant PDYN protein product. Three novel PDYNs expressed from spliced or truncated PDYN transcripts either lack a central segment but are still processed into dynorphins, or are translated into N-terminally truncated proteins. One truncated PDYN is located in the cell nucleus, suggesting a novel nonopioid function for this protein. The complexity of PDYN expression and diversity of its protein products may be relevant for diverse levels of plasticity in adaptive responses for the dynorphin system relevant for chronic pain.

General conclusions

Data generated in 2003-2007 within the project supported by AFA Forsäkring confirmed and further developed our initial observations on the critical role of the dynorphin system in maintenance of neuropathic pain, and functions of the UPS in the development and maintenance of neuropathic pain, as well as strong antinociceptive effects of proteasome inhibitors. The outcome of the study is a proof of principle that the UPS is a target for pharmacotherapy of neuropathic pain. In addition, the critical role of the dynorphin system which generates endogenous pathological agents, dynorphins and mechanisms of non-opioid and opioid actions have been better characterized and understood.

Published and submitted papers supported by the AFA Forsäkring grant

19. Hugonin L, Vukojevic V. Bakalkin G. and Graslund A. Calcium influx into phospholipid vesicles caused by dynorphin neuropeptides. Biochem. Biophys. Acta In Press. 18. Ossipov M.H., Bazov I., Gardell L.R., Kowal J., Yakovleva T., Usynin I., Ekstrom T.J., Porreca F. and Bakalkin G. Control of chronic pain by the ubiquitin proteasome system in the spinal cord. J Neurosci. 2007, 27, 8226-8237. 17. Nazira EH, Kelps KA, Zou SP, Zhao TY, Bruce-Keller AJ, Yakovleva T, Bakalkin G, Knapp PE, and Hauser KF. Convergent [Ca2+]i PI3K, and NF-ĸB Signals Underlie Morphine and HIV-1 Tat Synergistic Increases in MCP-1 and IL-6 Release by Astrocytes. Submitted. 16. Adjan V.V., Hauser K.F., Bakalkin G., Yakovleva T., Gharibyan A., Scheff S.W., Knapp P.E. Caspase-3 activity is reduced after spinal cord injury in mice lacking dynorphin: 7 Professor Tomas Ekström and Docent Georgy Bakalkin 2/9/2009 ______Differential effects on glia and neurons. Neuroscience 2007 148(3): 724-36. 15. Yakovleva T., Bazov I., Cebers G., Marinova Z., Ahmed A., Vlaskovska M., Hara Y., Johansson B., Hochgeschwender U., Singh I.N., Bruce-Keller A.J., Hurd Y.L., Terenius L., Ekström T.J., Hauser K.F., Pickel V.M. and Bakalkin G. Prodynorphin is located and processed to dynorphins in axon terminals and dendrites. FASEB J. 20: 2124-2126. Epub 2006 Sep 11. 14. Yakovleva T., Marinova Z., Kuzmin A., Seidah N.G., Haroutunian V., Terenius L. and Bakalkin G. Dysregulation of dynorphins in Alzheimer disease. Neurobiol. Aging. 2006 Aug 14; [Epub ahead of print]. 13. Kuzmin A, Kreek M.J., Bakalkin G. and Liljequist S. The /orphanin FQ receptor agonist Ro 64-6198 reduces alcohol self-administration and prevents relapse to alcohol. Neuropsychopharmacology, 2006 Jul 26; [Epub ahead of print]. 12. Hugonin L., Vukojević V., Bakalkin G. and Gräslund A. Membrane leakage induced by dynorphins. FEBS Lett. 2006 580, 3201-3205. 11. Karimi M., Johansson S., Stach D., Corcoran M., Grandir D., Schalling M., Bakalkin G., Lyko F., Larsson C., and Ekström T. LUMA (LUminometric Methylation Assay) - A high throughput method to the analysis of genomic DNA methylation. Exptl. Cell Res. 2006, 312, 1989-1995. 10. Drakenberg K., Nikoshkov A., Horváth M.Cs., Gharibyan A., Fagergren P., Saarelainen K., Rahman S., Nylander I., Bakalkin G., Rajs J., Keller Eva., Hurd Y.L. Mu opioid receptor A118G polymorphism in association with striatal opioid neuropeptide gene expression in abusers. Proc. Natl. Acad. Sci. USA 2006, 103, 7883–7888. 9. Woods A.S., Kaminski R.M., Wang Y., Oz M., Hauser K.F., Goody R.J., Wang H.-Y. J., Zeitz P., Zeitz K.P., Zolkowska D., Schepers R., Chang C.-f., Nold M., Bakalkin G., Basbaum A.I. and Shippenberg T.S. Novel decoy peptides that scavenge dynorphin prevent ischemic brain injury and NMDA-receptor mediated neurotoxicity. J. Proteome Res. 2006, 5, 1017- 1023. 8. Hara Y., Yakovleva T., Bakalkin G. and Pickel V.M. Subcellular distributions of prodynorphin and dopamine d1 receptors in the rat nucleus accumbens shell. Synapse 2006, 60: 1-19. 7. Merg F., Filliol D., Usynin I., Bazov I., Bark N., Hurd Y.L., Yakovleva T., Kieffer B.L. and Bakalkin G. Big dynorphin as a putative endogenous ligand for the kappa-opioid receptor. J. Neurochem. 2006, 97, 292–301. 6. Kuzmin A., Madjid N., Terenius L., Ogren S.O. and Bakalkin G. Big dynorphin, a prodynorphin-derived peptide produces NMDA receptor-mediated effects on memory, anxiolytic-like and locomotor behavior in mice. Neuropsychopharmacology, 2006 31: 1928-1937. 5. Nikoshkov A., Hurd Y.L., Yakovleva T., Marinova Z., Pasikova N., Cebers G., Terenius L., and Bakalkin G. Prodynorphin transcripts and proteins differentially expressed and regulated in the adult human brain. FASEB J. 19: 1543-1545, 2005. 4. Marinova Z., Vukojević V., Surcheva S., Yakovleva T., Cebers G., Pramanik A., Pasikova N., Usynin I., Hugonin L., Weijie F., Hallberg M., Hirschberg D., Bergman T., Hauser K.F., Aldrich J.V., Gräslund A., Terenius L. and Bakalkin G. Translocation of dynorphin

8 Professor Tomas Ekström and Docent Georgy Bakalkin 2/9/2009 ______neuropeptides across the plasma membrane: a putative mechanism of signal transmission. J. Biol. Chem. 280: 26360-26370, 2005. 3. Nguyen X.V., Masse J., Kumar A., Vijitruth R., Kulik C., Liu M., Choi D.Y., Foster T.C., Usynin I., Bakalkin G. and Bing G. Prodynorphin knockout mice demonstrate diminished age-associated impairment in spatial water maze performance. Behav Brain Res. 161, 254- 262, 2005. 2. Vukojević V., Pramanik A., Yakovleva T., Rigler R., Terenius L. and Bakalkin G. Study of Molecular Events in Cells by Fluorescence Correlation Spectroscopy. Cell. Mol. Life Sci. 62, 535-550, 2005. 1. Tan-No K., Takahashi H., Nakagawasai O., Niijima F., Sato T., Satoh S., Sakurada S., Marinova Z., Yakovleva T., Bakalkin G., Terenius L., and Tadano T. Pronociceptive role of dynorphins in uninjured animals: N-ethylmaleimide-induced nociceptive behavior mediated through inhibition of dynorphin degradation. Pain 113, 301-309, 2005.

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