BACKGROUND: Chronic exposure of mammalian organism to morphine results in adaption to persistent high opioid tone through homeostatic adjustments. Our previous results indicated that in the frontal brain cortex (FBC) of rats exposed to morphine for 10 days, such a compensatory adjustment was detected as large up-regulation of adenylylcyclases I (8-fold) and II (2.5-fold). The other isoforms of AC (III-IX) were unchanged. Importantly, the increase of ACI and ACII was reversible as it disappeared after 20 days of morphine withdrawal. Changes of down-stream signaling molecules such as G proteins and adenylylcyclases should respond to and be preceded by primary changes proceeding at receptor level. Therefore in our present work, we addressed the problem of reversibility of the long-term morphine effects on μ-, δ- and κ-OR protein levels in FBC. METHODS: Rats were exposed to increasing doses of morphine (10-40 mg/kg) for 10 days and sacrificed either 24 h (group +M10) or 20 days (group +M10/-M20) after the last dose of morphine in parallel with control animals (groups -M10 and -M10/-M20). Post-nuclear supernatant (PNS) fraction was prepared from forebrain cortex, resolved by 1D-SDS-PAGE under non-dissociated (-DTT) and dissociated (+DTT) conditions, and analyzed for the content of μ-, δ- and κ-OR by immunoblotting with C- and N-terminus oriented antibodies. RESULTS: Significant down-regulation of δ-OR form exhibiting Mw ≈ 60 kDa was detected in PNS prepared from both (+M10) and (+M10/-M20) rats. However, the total immunoblot signals of μ-, δ- and κ-OR, respectively, were unchanged. Plasma membrane marker Na, K-ATPase, actin and GAPDH were unaffected by morphine in both types of PNS. Membrane-domain marker caveolin-1 and cholesterol level increased in (+M10) rats and this increase was reversed back to control level in (+M10/-M20) rats. CONCLUSIONS: In FBC, prolonged exposure of rats to morphine results in minor (δ-OR) or no change (μ- and κ-OR) of opioid receptor content. The reversible increases of caveolin-1 and cholesterol levels suggest participation of membrane domains in compensatory responses during opioid withdrawal. GENERAL SIGNIFICANCE: Analysis of reversibility of morphine effect on mammalian brain.

Department of Biochemistry Faculty of Science Charles University Prague Prague Czech Republic

Department of Biomathematics Institute of Physiology of the Czech Academy of Sciences Prague Czech Republic

See more in PubMed

Kieffer BL. Opioids: first lessons from knockout mice. Trends Pharmacol Sci. 1999;20: 19–26. PubMed

Williams JT, Christie MJ, Manzoni O. Cellular and synaptic adaptations mediating opioid dependence. Physiol Rev. 2001;81: 299–343. PubMed

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

Williams JT, Ingram SL, Henderson G, Chavkin C, von Zastrow M, Schulz S, et al. Regulation of μ-opioid receptors: desensitization, phosphorylation, internalization, and tolerance. Pharmacol Rev. 2013;65: 223–254. doi: 10.1124/pr.112.005942 PubMed DOI PMC

King T, Ossipov MH, Vanderah TW, Porreca F, Lai J. Is paradoxical pain induced by sustained opioid exposure an underlying mechanism of opioid antinociceptive tolerance? Neurosignals. 2005;14: 194–205. doi: 10.1159/000087658 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

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

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

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. 2011;1810: 1220–1229. doi: 10.1016/j.bbagen.2011.09.017 PubMed DOI

Ammer H, Schulz R. Enhanced stimulatory adenylyl cyclase signaling during opioid dependence is associated with a reduction in palmitoylated Gs alpha. Mol Pharmacol. 1997;52: 993–999. PubMed

Ammer H, Schulz R. Adenylyl cyclase supersensitivity in opioid-withdrawn NG108-115 hybrid cells requires Gs but is not mediated by Gs alpha subunit. J Pharmacol Exp Ther. 1998;286: 855–862. PubMed

Avidor-Reiss T, Bayewitch M, Levy R, Matus-Leibovitch N, Nevo I, Vogel Z. Adenylylcyclase supersensitization in mu-opioid receptor-transfected Chinese hamster ovary cells following chronic opioid treatment. J Biol Chem. 1995;270: 29732–29738. PubMed

Avidor-Reiss T, Nevo I, Levy R, Pfeuffer T, Vogel Z. Chronic opioid treatment induces adenylyl cyclase V superactivation. Involvement of Gbetagamma. J Biol Chem. 1996;271: 21309–21315. PubMed

Varga EV, Rubenzik MK, Stropova D, Sugiyama M, Grife V, Hruby VJ, Rice KC, Roeske WR, Yamamura HI. Converging protein kinase pathways mediate adenylyl cyclase superactivation upon chronic delta-opioid agonist treatment. J Pharmacol Exp Ther. 2003;306: 109–15. doi: 10.1124/jpet.103.049643 PubMed DOI

Ujcikova H, Vosahlikova M, Roubalova L, Svoboda P. Proteomic analysis of protein composition of rat forebrain cortex exposed to morphine for 10 days; comparison with animals exposed to morphine and subsequently nurtured for 20 days in the absence of this drug. J Proteomics. 2016;145: 11–23. doi: 10.1016/j.jprot.2016.02.019 PubMed DOI

Ujcikova H, Eckhardt A, Kagan D, Roubalova L, Svoboda P. Proteomic analysis of post-nuclear supernatant and percoll-purified membranes prepared from brain cortex of rats exposed to increasing doses of morphine. Proteome Sci. 2014;12: 11 doi: 10.1186/1477-5956-12-11 PubMed DOI PMC

Bourova L, Stohr J, Lisy V, Rudajev V, Novotny J, Svoboda P. Isolation of plasma membrane compartments from rat brain cortex; detection of agonist-stimulated G protein activity. Med Sci Monit. 2009;15: BR111–BR122. PubMed

Dlouha K, Kagan D, Roubalova L, Ujcikova H, Svoboda P. Plasma membrane density of GABAB-R1a, GABAB-R1b, GABA-R2 and trimeric G-proteins in the course of postnatal development of rat brain cortex. Physiol Res. 2013;62: 547–559. PubMed

Svoboda P, Amler E, Teisinger J. Different sensitivity of ATP +Mg+Na (I) and Pi+Mg (II) dependent types of oubain binding to phospholipase A2. J Membr Biol. 1988;104: 211–221. PubMed

Ritchie T, Noble EP. [3H]naloxone binding in the human brain: alcoholism and Taq I A D2 dopamine receptor polymorphism. Brain Research. 1996;718: 193–197. PubMed

Ko MCH, Lee H, Harrison C, Clark MJ, Song HF, Naughton NN, et al. Studies of μ-, δ- and κ-opioid receptor density and G protein activation in the cortex and thalamus of monkeys. J Pharmacol Exp Ther. 2003;306: 179–186. doi: 10.1124/jpet.103.050625 PubMed DOI

De Vries TJ, Tjon Tien Ril GHK, Van der Laan JW, Mulder AH, Schoffelmeer ANM. Chronic exposure to morphine and naltrexone induces changes in catecholaminergic neurotransmission in rat brain without altering μ-opioid receptor sensitivity. Life Sci. 1993;52: 1685–1693. PubMed

Garzon J, Juarros JL, Castro MA, Sanchez-Blazquez P. Antibodies to the cloned μ-opioid receptor detect various molecular weight forms in areas of mouse brain. Mol Pharmacol. 1995;47: 738–744. PubMed

Huang P, Liu-Chen LY. Detecting the mu opioid receptor in brain following SDS-PAGE with multiple approaches. Front Biosci (Elite Ed). 2009;1: 220–227. PubMed PMC

Kasai S, Yamamoto H, Kamegaya E, Uhl GR, Sora I, Watanabe M, et al. Quantitative detection of μ opioid receptor: western blot analyses using μ opioid receptor knockout mice. Curr Neuropharmacol. 2011;9: 219–222. doi: 10.2174/157015911795016921 PubMed DOI PMC

Cvejic S, Devi LA. Dimerization of the δ opioid receptor: implication for a role in receptor internalization. J Biol Chem. 1997;272: 26959–26964. PubMed

Devi LA. Heterodimerization of G-protein-coupled receptors: pharmacology, signaling and trafficking. Trends Pharmacol Sci. 2001;22: 532–537. PubMed

Brejchova J, Sykora J, Dlouha K, Roubalova L, Ostasov P, Vosahlikova M, et al. Fluorescence spectroscopy studies of HEK293 cells expressing DOR-Gi1α fusion protein; the effect of cholesterol depletion. Biochim Biophys Acta. 2011;1808: 2819–2829. doi: 10.1016/j.bbamem.2011.08.010 PubMed DOI

Brejchova J, Vosahlikova M, Roubalova L, Parenti M, Mauri M, Chernyavskiy O, et al. Plasma membrane cholesterol level and agonist-induced internalization of δ-opioid receptors; colocalization study with intracellular membrane markers of Rab family. J Bioenerg Biomembr. 2016;48: 375–396. doi: 10.1007/s10863-016-9667-7 PubMed DOI

Ostasov P, Bourova L, Hejnova L, Novotny J, Svoboda P. Disruption of the plasma membrane integrity by cholesterol depletion impairs effectiveness of TRH receptormediated signal transduction via G(q)/G(11)alpha proteins. J Recept Signal Transduct Res. 2007;27: 335–352. doi: 10.1080/10799890701684142 PubMed DOI

Ostasov P, Krusek J, Durchankova D, Svoboda P, Novotny J. Ca2+ responses to thyrotropin-releasing hormone and angiotensin II: the role of plasma membrane integrity and effect of G(11)alpha protein overexpression on homologous and heterologous desensitization. Cell Biochem Funct. 2008;26: 264–274. doi: 10.1002/cbf.1466 PubMed DOI

Ostasov P, Sykora J, Brechova J, Olzynska A, Hof M, Svoboda P. FLIM studies of 22-and 25-NBD-cholesterol in living HEK293 cells: plasma membrane change induced by cholesterol depletion. Chem Phys Lipids. 2013;167: 62–69. doi: 10.1016/j.chemphyslip.2013.02.006 PubMed DOI

Brejchova J, Sykora J, Ostasov P, Merta L, Roubalova L, Janacek J, et al. TRH-receptor mobility and function in intact and cholesterol-depleted plasma membrane of HEK293 cells stably expressing TRH-R-eGFP. Biochim Biophys Acta. 2015;1848: 781–796. doi: 10.1016/j.bbamem.2014.11.029 PubMed DOI

Zhao H, Loh HH, Law PY. Adenylylcyclase super-activation induced by the long-term treatment with opioid agonist is dependent on receptor localized within lipid rafts and is independent of receptor internalization. Mol Pharmacol. 2006;69: 1421–1432. doi: 10.1124/mol.105.020024 PubMed DOI

Zheng H, Chu J, Qiu Y, Loh HH, Law PY. Agonist-selective signaling is determined by the receptor location within the membrane proteins. Proc Natl Acad Sci USA. 2008;105: 9421–9426. doi: 10.1073/pnas.0802253105 PubMed DOI PMC

Huang P, Xu W, Yoon SI, Chen C, Chong PL, Unterwald EM, et al. Agonist treatment did not affect association of mu opioid receptors with lipid rafts and cholesterol reduction had opposite effects on the receptor-mediated signaling in rat brain and CHO cells. Brain Res. 2007. a;1184: 46–56. doi: 10.1016/j.brainres.2007.09.096 PubMed DOI PMC

Huang P, Xu W, Yoon SI, Chen CG, Chong PLG, Liu-Chen LY. Cholesterol reduction by methyl-beta-cyclodextrin attenuates the delta opioid receptor-mediated signaling in neuronal cells but enhances it in non-neuronal cells. Biochem Pharmacol. 2007. b;73: 534–549. doi: 10.1016/j.bcp.2006.10.032 PubMed DOI PMC

Xu W, Yoon SI, Huang P, Wang YL, Chen CG, Chong PLG, et al. Localization of the kappa opioid receptor in lipid rafts. J Pharmacol Exp Ther. 2006;317: 1295–1306. doi: 10.1124/jpet.105.099507 PubMed DOI

Qiu Y, Wang Y, Law PY, Chen HZ, Loh HH. Cholesterol regulates micro-opioid receptor-induced β-arrestin 2 translocation to membrane lipid rafts. Mol Pharmacol. 2011;80: 210–218. doi: 10.1124/mol.110.070870 PubMed DOI PMC

Zheng H, Zou H, Liu X, Chu J, Zhou Y, Loh HH, Law PY. Cholesterol level influences opioid signaling in cell models and analgesia in mice and humans. J Lipid Res. 2012;53: 1153–1162. doi: 10.1194/jlr.M024455 PubMed DOI PMC

Pan L, Xu J, Yu R, Xu MM, Pan YX, Pasternak GW. Identification and characterization of six new alternatively spliced variants of the human μ opioid receptor gene, Oprm. Neuroscience. 2005;133: 209–220. doi: 10.1016/j.neuroscience.2004.12.033 PubMed DOI

Castelli MP, Melis M, Mameli M, Fadda P, Diaz G, Gessa GL. Chronic morphine and naltrexone fail to modify μ-opioid receptor mRNA levels in the rat brain. Brain Res Mol Brain Res. 1997;45: 149–153. PubMed

Wang C, Shu SY, Guo Z, Cai YF, Bao X, Zeng C, et al. Immunohistochemical localization of mu opioid receptor in the marginal division with comparison to patches in the neostriatum of the rat brain. J Biomed Sci. 2011;18: 34 doi: 10.1186/1423-0127-18-34 PubMed DOI PMC

Xu Y, Gu Y, Xu GY, Wu P, Li GW, Huang LYM. Adeno-associated viral transfer of opioid receptor gene to primary sensory neurons: a strategy to increase opioid anti-nociception. Proc Natl Acad Sci USA. 2003;100: 6204–6209. doi: 10.1073/pnas.0930324100 PubMed DOI PMC

Kosten TR, George TP. The neurobiology of opioid dependence: implications for treatment. Sci Pract Perspect. 2002;1: 13–20. PubMed PMC

Rehni AK, Jaggi AS, Singh N. Opioid withdrawal syndrome: emerging concepts and novel therapeutic targes. CNS Neurol Disord Drug Targets. 2013;12: 112–125. PubMed

Bhalla S, Andurkar SV, Gulati A. Neurobiology of opioid withdrawal: role of the endothelin system. Life Sci. 2016;159: 34–42. doi: 10.1016/j.lfs.2016.01.016 PubMed DOI

Stinus L, Caille S, Koob GF. Opiate withdrawal-induced place aversion lasts for up to 16 weeks. Psychopharmacology 2000;149: 115–120. PubMed

Stinus L, Cador M, Caille S. Repeated episodes of heroin cause enduring alterations of circadian activity in protracted abstinence. Brain Sci. 2012;2: 421–433. doi: 10.3390/brainsci2030421 PubMed DOI PMC

Erbs E, Faget L, Ceredig RA, Matifas A, Vonesch JL, Kieffer BL, et al. Impact of chronic morphine on delta opioid receptor-expressing neurons in the mouse hippocampus. Neuroscience. 2016;313: 46–56. doi: 10.1016/j.neuroscience.2015.10.022 PubMed DOI PMC

Lalanne L, Ayranci G, Kieffer BL, Lutz PE. The kappa opioid receptor: from addiction to depression, and back. Front Psychiatry. 2014;5: 1–17. PubMed PMC

Ott D, Frischknecht R, Plückthun A. Construction and characterization of a kappa opioid receptor devoid of all free cysteines. Protein Eng Des Sel. 2004;17: 37–48. doi: 10.1093/protein/gzh004 PubMed DOI

Nascimento AIR, Ferreira HS, Saraiva RM, Almeida TS, Fregoneze JB. Central kappa opioid receptors modulate salt appetite in rats. Physiol Behav. 2012;106: 506–514. doi: 10.1016/j.physbeh.2012.03.028 PubMed DOI

Shippenberg TS. The dynorphin/kappa opioid receptor system: a new target for the treatment of addiction and affective disorders? Neuropsychopharmacology. 2009;34: 247. PubMed

Vanderah TW. Delta and kappa opioid receptors as suitable drug targets for pain. Clin J Pain. 2010;89: 1379–1412. PubMed

Pradhan AA, Befort K, Nozaki C, Gavériaux-Ruff C, Kieffer BL. The delta opioid receptor: an evolving target for the treatment of brain disorders. Trends Pharmacol Sci. 2011;32: 581–590. doi: 10.1016/j.tips.2011.06.008 PubMed DOI PMC

Simonds K, Ikonen E. Functional rafts in cell membranes. Nature. 1997;387: 569–572. doi: 10.1038/42408 PubMed DOI

Anderson RG. The caveolae membrane system. Annu Rev Biochem. 1998;67: 199–225. doi: 10.1146/annurev.biochem.67.1.199 PubMed DOI

Ostrom RS, Insel PA. The evolving role of lipid rafts and caveolae in G protein-coupled receptor signaling: implications for molecular pharmacology. Br J Pharmacol. 2004;143: 235–245. doi: 10.1038/sj.bjp.0705930 PubMed DOI PMC

Allen JA, Halverson-Tamboli RA, Rasenick MM. Lipid raft microdomains and neutrotransmitter signalling. Nat Rev Neurosci. 2007;8: 128–140. doi: 10.1038/nrn2059 PubMed DOI

Proteomic analysis of protein composition of rat hippocampus exposed to morphine for 10 days; comparison with animals after 20 days of morphine withdrawal

The Impact of Morphine on the Characteristics and Function Properties of Human Mesenchymal Stem Cells