BACKGROUND: Proteomic analysis was performed in post-nuclear supernatant (PNS) and Percoll-purified membranes (PM) prepared from fore brain cortex of rats exposed to increasing doses of morphine (10-50 mg/kg) for 10 days. RESULTS: In PNS, the 10 up (↑)- or down (↓)-regulated proteins exhibiting the largest morphine-induced change were selected, excised manually from the gel and identified by MALDI-TOF MS/MS: 1-(gi|148747414, Guanine deaminase), ↑2.5×; 2-(gi|17105370, Vacuolar-type proton ATP subunit B, brain isoform), ↑2.6×; 3-(gi|1352384, Protein disulfide-isomerase A3), ↑3.4×; 4-(gi|40254595, Dihydropyrimidinase-related protein 2), ↑3.6×; 5-(gi|149054470, N-ethylmaleimide sensitive fusion protein, isoform CRAa), ↑2.0×; 6-(gi|42476181, Malate dehydrogenase, mitochondrial precursor), ↑1.4×; 7-(gi|62653546, Glyceraldehyde-3-phosphate dehydrogenase), ↑1.6×; 8-(gi|202837, Aldolase A), ↑1.3×; 9-(gi|31542401, Creatine kinase B-type), ↓0.86×; 10-(gi|40538860, Aconitate hydratase, mitochondrial precursor), ↑1.3×. The identified proteins were of cytoplasmic (1, 4, 5, 7, 9), cell membrane (2), endoplasmic reticulum (3) and mitochondrial (6, 8, 10) origin and 9 of them were significantly increased, 1.3-3.6×. The 4 out of 9 up-regulated proteins (4, 6, 7, 10) were described as functionally related to oxidative stress; the 2 proteins participate in genesis of apoptotic cell death.In PM, the 18 up (↑)- or down (↓)-regulated proteins were identified by LC-MS/MS and were of plasma membrane [Brain acid soluble protein, ↓2.1×; trimeric Gβ subunit, ↓2.0x], myelin membrane [MBP, ↓2.5×], cytoplasmic [Internexin, ↑5.2×; DPYL2, ↑4.9×; Ubiquitin hydrolase, ↓2.0×; 60S ribosomal protein, ↑2.7×; KCRB, ↓2.6×; Sirtuin-2, ↑2.5×; Peroxiredoxin-2, ↑2.2×; Septin-11, ↑2.2×; TERA, ↑2.1×; SYUA, ↑2.0×; Coronin-1A, ↓5.4×] and mitochondrial [Glutamate dehydrogenase 1, ↑2.7×; SCOT1, ↑2.2×; Prohibitin, ↑2.2×; Aspartate aminotransferase, ↓2.2×] origin. Surprisingly, the immunoblot analysis of the same PM resolved by 2D-ELFO indicated that the "active", morphine-induced pool of Gβ subunits represented just a minor fraction of the total signal of Gβ which was decreased 1.2x only. The dominant signal of Gβ was unchanged. CONCLUSION: Brain cortex of rats exposed to increasing doses of morphine is far from being adapted. Significant up-regulation of proteins functionally related to oxidative stress and apoptosis suggests a major change of energy metabolism resulting in the state of severe brain cell "discomfort" or even death.

Laboratories of Biochemistry of Membrane Receptors Institute of Physiology v v i Academy of Sciences of the Czech Republic Videnska 1083 Prague 4 14220 Czech Republic

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Contet C, Kieffer BL, Befort K. Mu opioid receptor: a gateway to drug addiction. Curr Opin Neurobiol. 2004;14:370–378. doi: 10.1016/j.conb.2004.05.005. PubMed DOI

Preston KL. Drug abstinence effects: opioids. Br J Addict. 1991;86:1641–1646. doi: 10.1111/j.1360-0443.1991.tb01759.x. PubMed DOI

Connor M, Christie MD. Opioid receptor signalling mechanisms. Clin Exp Pharmacol Physiol. 1999;26:493–499. doi: 10.1046/j.1440-1681.1999.03049.x. PubMed DOI

Law PY, Wong YH, Loh HH. Molecular mechanisms and regulation of opioid receptor signaling. Annu Rev Pharmacol Toxicol. 2000;40:389–430. doi: 10.1146/annurev.pharmtox.40.1.389. PubMed DOI

Law PY, Loh HH, Wei LN. Insights into the receptor transcription and signaling: implications in opioid tolerance and dependence. Neuropharmacology. 2004;47:300–311. PubMed

Robinson TE, Kolb B. Morphine alters the structure of neurons in the nucleus accumbens and neocortex of rats. Synapse. 1999;33:160–162. doi: 10.1002/(SICI)1098-2396(199908)33:2<160::AID-SYN6>3.0.CO;2-S. PubMed DOI

Li KW, Jimenez CR, van der Schors RC, Hornshaw MP, Schoffelmeer ANM, Smit AB. Intermittent administration of morphine alters protein expression in rat nucleus accumbens. Proteomics. 2006;6:2003–2008. doi: 10.1002/pmic.200500045. PubMed DOI

Kim SY, Chudapongse N, Lee SM, Levin MC, Oh JT, Park HJ, Ho IK. Proteomic analysis of phosphotyrosyl proteins in morphine-dependent rat brains. Brain Res Mol Brain Res. 2005;133:58–70. doi: 10.1016/j.molbrainres.2004.09.018. PubMed DOI

Miller AL, Hawkins RA, Harris RL, Veech RL. The effects of acute and chronic morphine treatment and of morphine withdrawal on rat brain in vivo. Biochem J. 1972;129:463–469. PubMed PMC

Li Q, Zhao X, Zhong LJ, Yang HY, Wang Q, Pu XP. Effects of chronic morphine treatment on protein expression in rat dorsal root ganglia. Eur J Pharmacol. 2009;612:21–28. doi: 10.1016/j.ejphar.2009.03.049. PubMed DOI

Bourova L, Vosahlikova M, Kagan D, Dlouha K, Novotny J, Svoboda P. Long-term adaptation to high doses of morphine causes desensitization of μ-OR- and δ-OR-stimulated G-protein response in forebrain cortex but does not decrease the amount of G-protein alpha subunit. Med Sci Monit. 2010;16:260–270. PubMed

Ujcikova H, Dlouha K, Roubalova L, Vosahlikova M, Kagan D, Svoboda P. Up-regulation of adenylylcyclases I and II induced by long-term adaptation of rats to morphine fades away 20 days after morphine withdrawal. Biochim Biophys Acta. 1810;2011:1220–1229. PubMed

Paletzki RF. Cloning and characterization of guanine deaminase from mouse and rat brain. Neuroscience. 2002;109:15–26. doi: 10.1016/S0306-4522(01)00352-9. PubMed DOI

Toei M, Saum R, Forgac M. Regulation and isoform function of the V-ATPases. Biochemistry. 2010;49:4715–4723. doi: 10.1021/bi100397s. PubMed DOI PMC

Tanaka S, Uehara T, Nomura Y. Up-regulation of protein-disulfide isomerase in response to hypoxia/brain ischemia and its protective effect against apoptotic cell death. J Bioch Chem. 2000;275:10388–10393. doi: 10.1074/jbc.275.14.10388. PubMed DOI

Conn KJ, Gao W, McKee A, Lan MS, Ullman MD, Eisenhauer PB, Fine RE, Wells JM. Identification of the protein disulfide isomerase family member PDIp in experimental Parkinson’s disease and Lewy body pathology. Brain Res. 2004;1022:164–172. doi: 10.1016/j.brainres.2004.07.026. PubMed DOI

Drabik A, Bierczynska-Krzysik A, Bodzon-Kulakowska A, Suder P, Kotlinska J, Silberring J. Proteomics in neurosciences. Mass Spectrom Rev. 2007;26:432–450. doi: 10.1002/mas.20131. PubMed DOI

Abul-Husn NS, Annangudi SP, Ma’ayan A, Ramos-Ortolaza DL, Stockton SD Jr, Gomes I, Sweedler JV, Devi LA. Chronic morphine alter the presynaptic protein profile: identification of novel molecular targets using proteomics and network analysis. PLoS One. 2011;6:e25535. doi: 10.1371/journal.pone.0025535. PubMed DOI PMC

Shi Q, Gibson GE. Up-regulation of the mitochondrial malate dehydrogenase by oxidative stress in mediated by miR-743a. J Neurochem. 2011;118:440–448. doi: 10.1111/j.1471-4159.2011.07333.x. PubMed DOI PMC

Chuang DM, Hough C, Senatorov VV. Glyceraldehyde-3-phosphate dehydrogenase, apoptosis, and neurodegenerative diseases. Annu Rev Pharmacol Toxicol. 2005;45:269–290. doi: 10.1146/annurev.pharmtox.45.120403.095902. PubMed DOI

Hwang NR, Yim SH, Kim YM, Jeong J, Song EJ, Lee Y, Choi S, Lee KJ. Oxidative modifications of glyceraldehyde-3-phophate dehydrogenase play a key role in its multiple cellular functions. Biochem J. 2009;423:253–264. doi: 10.1042/BJ20090854. PubMed DOI

Koppitz B, Vogel F, Mayr GW. Mammalian aldolases are isomer-selective high-affinity inositol polyphosphate binders. Eur J Biochem. 1986;161:421–433. doi: 10.1111/j.1432-1033.1986.tb10462.x. PubMed DOI

Baron CB, Tolan DR, Choi KH, Coburn RF. Aldolase A Ins(1,4,5)P3-binding domains as determined by site-directed mutagenesis. Biochem J. 1999;341:805–812. doi: 10.1042/0264-6021:3410805. PubMed DOI PMC

Hua LV, Green M, Warsh JJ, Li PP. Lithium regulation of aldolase A expression in the rat frontal cortex: identification by differential display. Biol Psychiatry. 2000;48:58–64. doi: 10.1016/S0006-3223(00)00824-6. PubMed DOI

Shen W, Willis D, Zhang Y, Schlattner U, Wallimann T, Molloy GR. Expression of creatine kinase isoenzyme genes during postnatal development of rat brain cerebellum:evidence for transcriptional regulation. Biochem J. 2002;367:369–380. doi: 10.1042/BJ20020709. PubMed DOI PMC

Perluigi M, Poon HF, Maragos W, Pierce WM, Klein JB, Calabrese V, Cini C, De Marco C, Butterfield DA. Proteomic analysis of protein expression and oxidative modification in R6/2 transgenic mice. Mol Cell Proteomics. 2005;4:1849–1861. doi: 10.1074/mcp.M500090-MCP200. PubMed DOI

Kashihara M, Miyata S, Kumanogoh H, Funatsu N, Matsunaga W, Kiyohara T, Sokawa Y, Maekawa S. Changes in the localization of NAP-22, a calmodulin binding membrane protein, during the development of neuronal polarity. Neurosci Res. 2000;37:315–325. doi: 10.1016/S0168-0102(00)00132-2. PubMed DOI

Okae H, Iwakura Y. Neural tube defects and impaired neural progenitor cell proliferation in Gβ1-deficient mice. Dev Dyn. 2010;239:1089–1101. doi: 10.1002/dvdy.22256. PubMed DOI

Sunahara RK, Taussig R. Isoforms of mammalian adenylylcyclase: multiplicities of signaling. Mol Interv. 2002;2:168–184. doi: 10.1124/mi.2.3.168. PubMed DOI

Wang HY, Burns LH. Gβγ that interacts with adenylyl cyclase in opioid tolerance originates from a Gs protein. J Neurobiol. 2006;12:1302–1310. PubMed

Perluigi M, Domenico FD, Giorgi A, Shininà ME, Coccia R, Cini C, Bellia F, Cambria MT, Cornelius C, Butterfield DA, Calabrese V. Redox proteomics in aging rat brain involvement of mitochondrial reduced glutathione status and mitochondrial protein oxidation in the aging process. J Neurosci Res. 2010;88:3498–3507. doi: 10.1002/jnr.22500. PubMed DOI

Kaplan MP, Chin SSM, Fliegner KH, Liem RKH. α-internexin, a novel neuronal intermediate filament protein, precedes the low molecular weight neurofilament protein (NF-L) in the developing rat brain. J Neurosci. 1990;10:2735–2748. PubMed PMC

Wu A, Ying Z, Gomez-Pinilla F. Oxidative stress modulates Sir2α in rat hippocampus and cerebral cortex. Eur J Neurosci. 2006;22:5213–5216. PubMed

Maries E, Dass B, Collier TJ, Kordower JH, Steece-Collier K. The role of α-synuclein in Parkinson’s disease: insights from animal models. Nat Rev Neurosci. 2003;4:727–738. doi: 10.1038/nrn1199. PubMed DOI

Rhee SG, Chae HZ, Kim K. Peroxiredoxins: a historical overview and speculative preview of novel mechanisms and emerging concepts in cell signaling. Free Radic Biol Med. 2005;38:1543–1552. doi: 10.1016/j.freeradbiomed.2005.02.026. PubMed DOI

Woodman PG. p97, a protein coping with multiple identities. J Cell Sci. 2003;116:4283–4290. doi: 10.1242/jcs.00817. PubMed DOI

Wang HF, Shih YT, Chen CY, Chao HW, Lee MJ, Hsueh YP. Valosin-containing protein and neurofibromin interact to regulate dendritic spine density. J Clin Invest. 2011;121:4820–4837. doi: 10.1172/JCI45677. PubMed DOI PMC

Cooper AJL. 13 N as a tracer for studying glutamate metabolism. Neurochem Int. 2011;59:456–464. doi: 10.1016/j.neuint.2010.11.011. PubMed DOI PMC

Ohnuki M, Takahashi N, Yamasaki M, Fukui T. Different localization in rat brain of the novel cytosolic ketone body-utilizing enzyme, acetoacetyl-CoA synthetase, as compared to succinyl-CoA:3 –oxoacid CoA-transferase. Biochim Biophys Acta. 2005;1729:147–153. doi: 10.1016/j.bbaexp.2005.05.006. PubMed DOI

Das SK, Hiran KR, Mukherjee S, Vasudevan DM. Oxidative stress is the primary event: effects of ethanol consumption in brain. Indian J Clin Biochem. 2007;22:99–104. doi: 10.1007/BF02912890. PubMed DOI PMC

Murphey RK, Godenschwege TA. New roles for ubiquitin in the assembly and function of neuronal circuits. Neuron. 2002;36:5–8. doi: 10.1016/S0896-6273(02)00943-1. PubMed DOI

Artal-Sanz M, Tavernarakis N. Prohibitin couples diapause signalling to mitochondrial metabolism during ageing in C.elegans. Nature. 2009;461:793–797. doi: 10.1038/nature08466. PubMed DOI

Merkwirth C, Langer T. Prohibitin function within mitochondria: essential roles for cell proliferation and cristae morphogenesis. Biochim Biophys Acta. 2009;1793:27–32. doi: 10.1016/j.bbamcr.2008.05.013. PubMed DOI

Mishra S, Ande SR, Nyomba BL. The role of prohibitin in cell signaling. FEBS J. 2010;277:3937–3946. doi: 10.1111/j.1742-4658.2010.07809.x. PubMed DOI

Zhou P, Qian L, D’Aurelio M, Cho S, Wang G, Manfredi G, Pickel V, Iadecola C. Prohibitin reduces mitochondrial free radical production and protects brain cells from different injury modalities. J Neurosci. 2012;32:583–592. doi: 10.1523/JNEUROSCI.2849-11.2012. PubMed DOI PMC

Tsujita K, Itoh T, Kondo A, Oyama M, Kozuka-Hata H, Irino Y, Hasegawa J, Takenawa T. Proteome of acidic phospholipid-binding proteins: spatial and temporal regulation of coronin 1A by phosphoinositides. J Biol Chem. 2010;285:6781–6789. doi: 10.1074/jbc.M109.057018. PubMed DOI PMC

Tada T, Simonetta A, Batterton M, Kinoshita M, Edbauer D, Sheng M. Role of septin cytoskeleton in spine morphogenesis and dendrite development in neurons. Curr Biol. 2007;17:1752–1758. doi: 10.1016/j.cub.2007.09.039. PubMed DOI PMC

Traudt CM, Tkac I, Ennis KM, Sutton LM, Mammel DM, Rao R. Postnatal morphine administration alters hippocampal development in rats. J Neurosci Res. 2012;90:307–314. doi: 10.1002/jnr.22750. PubMed DOI PMC

Plafker SM, Macara IG. Ribosomal protein L12 uses a distinct nuclear import pathway mediated by importin 11. Mol Cell Biol. 2002;22:1266–1275. doi: 10.1128/MCB.22.4.1266-1275.2002. PubMed DOI PMC

Filizola M, Devi LA. Structural biology: how opiod drugs bind to receptors. Nature. 2012;485:314–317. doi: 10.1038/485314a. PubMed DOI PMC

Manglik A, Kruse AC, Kobilka TS, Thian FS, Mathiesen JM, Sunahara RK, Pardo L, Weis WI, Kobilka BK, Granier S. Crystal structure of the μ-opioid receptor bound to a morphinan antagonist. Nature. 2012;485:321–326. doi: 10.1038/nature10954. PubMed DOI PMC

Sim LJ, Selley DE, Dworkin SI, Childers SR. Effects of chronic morphine administration on μ opioid receptor-stimulated [35S]GTPγS autoradiography in rat brain. J Neurosci. 1996;16:2684–2692. PubMed PMC

Maher CE, Martin TJ, Childers SR. Mechanisms of mu opioid receptor/G-protein desensitization in brain by chronic heroin administration. Life Sci. 2005;77:1140–1154. doi: 10.1016/j.lfs.2005.03.004. PubMed DOI

Sim-Selley LJ, Selley DE, Vogt LJ, Childers SR, Martin TJ. Chronic heroin self-administration desensitizes μ opioid receptor-activated G-proteins in specific regions of rat brain. J Neurosci. 2000;20:4555–4562. PubMed PMC

Bierczynska-Krzysik A, Bonar E, Drabik A, Noga M, Suder P, Dylag T, Dubin A, Kotlinska J, Silberring J. Rat brain proteome in morphine dependence. Neurochem Int. 2006;49:401–406. doi: 10.1016/j.neuint.2006.01.024. PubMed DOI

Bierczynska-Krzysik A, Pradeep John JP, Silberring J, Kotlinska J, Dylag T, Cabatic M, Lubec G. Proteomic analysis of rat cerebral cortex, hippocampus and striatum after exposure to morphine. Int J Mol Med. 2006;18:775–784. PubMed

Bodzon-Kułakowska A, Suder P, Mak P, Bierczynska-Krzysik A, Lubec G, Walczak B, Kotlinska J, Silberring J. Proteomic analysis of striatal neuronal cell cultures after morphine administration. J Sep Sci. 2009;32:1200–1210. doi: 10.1002/jssc.200800464. PubMed DOI

Drastichova Z, Bourova L, Hejnova L, Jedelsky P, Svoboda P, Novotny J. Protein alterations induced by long-term agonist treatment of HEK293 cells expressing thyrotropin-releasing hormone receptor and G11α protein. J Cell Biochem. 2010;109:255–264. PubMed

Kraus MA, Piper JM, Kornetsky C. Persistent increases in basal cerebral metabolic activity induced by morphine sensitization. Pharmacol Biochem Behav. 1997;57:89–100. doi: 10.1016/S0091-3057(96)00117-7. PubMed DOI

Magistretti PJ, Pellerin L. Cellular mechanisms of brain energy metabolism and their relevance to functional brain imaging. Philos Trans R Soc Lond B Biol Sci. 1999;354:1155–1163. doi: 10.1098/rstb.1999.0471. PubMed DOI PMC

Magistretti PJ, Allaman I. Glycogen: a Trojan horse for neurons. Nat Neurosci. 2007;10:1341–1342. doi: 10.1038/nn1107-1341. PubMed DOI

Guzman DC, Vazquez IE, Brizuela NO, Alvarez RG, Mejia GB, Garcia EH, Santamaria D, La Rosa De Apreza M, Olguin HJ. Assessment of oxidative damage induced by acute doses of morphine sulfate in postnatal and adult rat brain. Neurochem Res. 2006;31:549–554. doi: 10.1007/s11064-006-9053-7. PubMed DOI

Matousek P, Novotny J, Svoboda P. Resolution of G(s)alpha and G(q)alpha/G(11)alpha proteins in membrane domains by two-dimensional electrophoresis: the effect of long-term agonist stimulation. Physiol Res. 2004;53:295–303. PubMed

Matousek P, Novotny J, Rudajev V, Svoboda P. Prolonged agonist stimulation does not alter the protein composition of membrane domains in spite of dramatic changes induced in a specific signaling cascade. Cell Biochem Biophys. 2005;42:21–40. doi: 10.1385/CBB:42:1:021. PubMed DOI

Moravcova Z, Rudajev V, Stohr J, Novotny J, Cerny J, Parenti M, Milligan G, Svoboda P. Long-term agonist stimulation of IP prostanoid receptor depletes the cognate G(s)alpha protein in membrane domains but does not change the receptor level. Biochim Biophys Acta. 2004;1691:51–65. doi: 10.1016/j.bbamcr.2003.12.004. PubMed DOI

Gharahdaghi F, Weinberg CR, Meagher DA, Imai BS, Mische SM. Mass spectrometric identification of proteins from silver-stained polyacrylamide gel: a method for the removal of silver ions to enhance sensitivity. Electrophoresis. 1999;20:601–605. doi: 10.1002/(SICI)1522-2683(19990301)20:3<601::AID-ELPS601>3.0.CO;2-6. PubMed DOI

Shevchenko A, Wilm M, Vorm O, Mann M. Mass spectrometric sequencing of proteins from silver-stained polyacrylamide gels. Anal Chem. 1996;68:850–858. doi: 10.1021/ac950914h. PubMed DOI

Sinha P, Poland J, Schnölzer M, Rabilloud T. A new silver staining apparatus and procedure for matrix-assisted laser desorption/ionization-time of flight analysis of proteins after two-dimensional electrophoresis. Proteomics. 2001;1:835–840. doi: 10.1002/1615-9861(200107)1:7<835::AID-PROT835>3.0.CO;2-2. PubMed DOI

Fountoulakis M, Takács MF, Berndt P, Langen H, Takács B. Enrichment of low abundance proteins of Escherichia coli by hydroxyapatite chromatography. Electrophoresis. 1999;20:2181–2195. doi: 10.1002/(SICI)1522-2683(19990801)20:11<2181::AID-ELPS2181>3.0.CO;2-Q. PubMed DOI

Shevchenko A, Tomas H, Havlis J, Olsen JV, Mann M. In-gel digestion for mass spectrometric characterization of proteins and proteomes. Nat Protoc. 2006;1:2856–2860. PubMed

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