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Acute and chronic effects of morphine and naloxone on the phosphorylation of neurofilament-H proteins in the rat brain
Neuroscience Letters 304 (2001) 37±40
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Acute and chronic effects of morphine and naloxone on the phosphorylation of neuro®lament-H proteins in the rat brain Philippe E. Jaquet a, Marcel Ferrer-AlcoÂn a, Pedro Ventayol b, Jose GuimoÂn a, JesuÂs A. GarcõÂa-Sevilla a,b,* a
Clinical Research Unit, Department of Psychiatry, University of Geneva, HUG Belle-IdeÂe (Le SaleÁve), 2 Chemin du Petit-Bel-Air, CH-1225 CheÃne-Bourg, Switzerland b Laboratory of Neuropharmacology, Associate Unit of the Institute Cajal/ CSIC, Department of Biology, University of the Balearic Islands, Cra. Valldemossa Km 7.5, E-07071 Palma de Mallorca, Spain Received 30 January 2001; received in revised form 14 March 2001; accepted 14 March 2001
Abstract Increased amounts of phosphorylated neuro®laments (pNF-H and pNF-M) are found in postmortem brains of opioid addicts. Because of the potential relevance of aberrant pNF in opioid addiction (alterations of neuronal cytoskeleton and associated functions), the effects of opiate drugs on pNF-H were investigated in rat brain. Acute morphine (30 mg/kg, 2 h) induced a marked increase in the immunodensity of pNF-H in the cerebral cortex (93%). Chronic morphine (10±100 mg/kg for 5 days) followed by opiate withdrawal resulted in a time-dependent decline in pNF-H (induction of tolerance). Thus, 2 h after the last dose of morphine, the abundance of pNF-H was still increased (27%), which was followed (6±24 h) by down-regulation of pNF-H (5% increase at 6 h; 5% decrease at 12 h, and 29% decrease at 24 h). The acute (10 mg/kg for 2 h) and chronic (2 £ 10 mg/kg for 14 days) treatments with naloxone, an opioid receptor antagonist, did not alter pNF-H in the cerebral cortex, suggesting that the opioid receptors (probably the m-type) modulating the phosphorylation state of NF-H are not tonically activated by endogenous opioids. The results indicate that morphine addiction is associated with an aberrant hyperphophorylation of NF-H in the rat brain. q 2001 Published by Elsevier Science Ireland Ltd. Keywords: Morphine; Naloxone; Opioid addiction; Neuro®lament-H; Phosphorylation; Rat brain
The neuro®lament (NF-H, NF-M and NF-L) proteins (neuronal intermediate ®laments) are the major elements of the cytoskeleton in mature neurones and abundant throughout the perikarya, axons and dendrites [10,14]. Phosphorylation of NF appears to be a major mechanism for NF assembly/disassembly, a process which is essential for the integrity of the neuronal cytoskeleton and associated functions (e.g. axonal transport, axonal plasticity and neuronal morphology [10,12,13,19]. Chronic treatment of rats with morphine has been associated with decreases in the immunodensities of nonphosphorylated NF (mainly NF-L) proteins in the ventral tegmental area [1] and in the frontal cortex [3], two brain regions which are targets for the chronic effects of opioid drugs [5,16,17]. Recently, the immunodensities of nonphosphorylated NF-H and NF-L proteins also were shown to be * Corresponding author. Tel.: 141-22-3055756; fax: 141-223055799. E-mail address: [email protected] (J.A. GarcõÂaSevilla).
decreased in postmortem brains (prefrontal cortex) of chronic opioid abusers [6,7]. In marked contrast, increased immunodensities of phosphorylated NF-H and NF-M were found in the same brains of opioid addicts [6]. These reductions in total NF proteins and the aberrant hyperphosphorylation of NF-H in brains of opioid addicts could play a major role in the cellular mechanisms of opioid addiction [6,11,16]. The increases in phosphorylated NF proteins in brains of opioid addicts [6] could be related to both the chronic effect of the opioid and the ®nal acute effect of the deadly opioid overdose (mainly heroin). Because of the potential relevance of NF phosphorylation in human opioid addiction, this study was designed to assess the in¯uence of the opioid system (acute and chronic effects of morphine and naloxone) on the phosphorylation of NF-H in the rat brain. Male Sprague±Dawley rats (250±300 g) were used. The animals were housed under controlled environmental conditions (228C, 70% humidity, and 12-h light/dark cycle) with free access to food and water. For the acute treatments, the
0304-3940/01/$ - see front matter q 2001 Published by Elsevier Science Ireland Ltd. PII: S03 04 - 394 0( 0 1) 01 72 9- 3
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P.E. Jaquet et al. / Neuroscience Letters 304 (2001) 37±40
rats received a single intraperitoneal (i.p.) injection of morphine (30 mg/kg i.p. for 2 h) or naloxone (10 mg/kg i.p. for 2 h). For the chronic treatment with morphine, the rats were injected i.p. three times daily during 5 days with increasing doses of the opiate (10±100 mg/kg) [5,20], and the animals were sacri®ced 2, 6, 12 or 24 h after the last dose. In another series of experiments, rats were treated with heroin (10±30 mg/kg for 5 days) [20] and the animals were killed 24 h after the last dose. For the chronic treatment with naloxone, the rats received 10 mg/kg i.p. every 12 h during 14 days, and the animals were sacri®ced 2 h after the last dose. Control rats received 0.9% saline vehicle i.p. (1 ml/kg) at the indicated treatment times. The rats were killed by decapitation and specimens of cerebral cortex were dissected and stored at 2808C until use. Morphine HCl and heroin HCl were from UnioÂn-QuõÂmico-FarmaceÂutica S.A.E., Madrid, Spain; and naloxone HCl was from Endo Laboratories, New York, USA. These experiments in rats were performed according to the guidelines of the University of the Balearic Islands, Spain. Preparation of cerebral membranes (containing the cytoskeleton) and quantitation of NF proteins (Western blotting) were assessed as described previously [6,20]. About 100 mg of cerebral frontal cortex was homogenized (1:20, w/v) in 40 mM Tris buffer (pH 7.5) containing 1% Triton X-100, 1 mM EDTA, 1 mM MgCl2, 5 mM NaCl, and the protease inhibitors phenylmethylsulfonyl ¯uoride (1 mM) and leupeptin (40 mg/ml). The samples were centrifuged at 40 000 £ g for 45 min, and then 100 ml of the resulting supernatant (containing the cytoskeleton) was mixed with an equal volume of electrophoresis loading buffer [20], which was then boiled (denatured) and stored at 2208C until use. Proteins were determined by the method of Bradford [4]. 40 mg protein of each brain sample was submitted to SDS-PAGE on 15-well (6 £ 8 cm minigels, 1.0 mm thick) 7% polyacrylamide gels. Proteins were electrophoretically transferred to nitrocellulose membranes (immunoblotting) that were incubated (1 h) in phosphate-buffered saline containing 5% nonfat dry milk, 0.2% Tween 20 and 0.5% bovine serum albumine (blocking solution) [8]. Then the membranes were incubated overnight at 48C in blocking solution containing the appropriate primary monoclonal antibody: phosphorylated NF-H (anti-NF 200 kDa, clone NE-14, Lot 056H4812, 1:1000 dilution, Sigma BioSciences, St. Louis, MO, USA; and antibody SMI-31, 1:5000 dilution, batch 13, Sternberger Monoclonals, Baltimore, MD, USA) and nonphosphorylated NF-L (anti-NF 68 kDa, clone NR-4, Lot 083H4811, 1:500 dilution, Sigma BioSciences). The secondary antibody, sheep anti-mouse IgG was incubated at 1:7,000 dilution in blocking solution for 2 h. Immunoreactivity was detected with an enhanced chemoluminescence (ECL) western blot detection system (Amersham, Buckinghamshire, UK), followed by exposure to Amersham Hyper®lm ECL for 1±10 min. The autoradiograms were quantitated by densitometric scanning (GS-700 Imaging Densitometer, Bio-Rad, Hercules, CA, USA) by measuring
the integrated optical density (IOD) of the immunoreactive bands. The amount of NF proteins in the prefrontal cortex of rats treated with opiates was compared in the same gel with that of control rats, which received saline solution. Prior to analyses, the linearity of protein concentration for Western blotting was ascertained by resolution of selected concentrations of protein (i.e., total protein loaded versus IOD units, consisting of six points of different protein content, 10 to 60 mg, resulting in linear relations) (data not shown). Experiments were performed by using 40 mg known to be within the linear range for immunolabeling of the target proteins. This quanti®cation procedure was assessed twice in different gels. Finally, percent changes in immunoreactivity with respect to control samples (100%) were calculated for each rat treated with morphine, heroin or naloxone in the two gels and the mean value used as a ®nal estimate. Results are expressed as the mean ^ SEM values. One-way analysis of variance (ANOVA), followed by Bonferroni's multiple comparison test, was used for the statistical evaluation. The level of signi®cance was chosen as P 0:05: Immunoreactive bands of 200 kDa and 68 kDa, corresponding respectively to the NF-H and NF-L cytoskeletal proteins [6,10,14], were found in the frontal cortex of rats after immunoblotting (Fig. 1). In preliminary experiments NF antibodies were tested for their speci®city for phosphorylated NF-H proteins. Antibodies NE-14 (Fig. 1A) and SMI-31 (data not shown) (see Ref. [6]) reacted speci®cally with phosphorylated epitopes of NF-H, because its enzymatic dephosphorylation with alkaline phosphatase completely abolished the immunoreactivity of the antibodies (see Fig. 1A). The immunoreactivity of phospho-speci®c antibodies against NF-H re¯ects the phosphorylationdependent alteration of NF organization [12,13]. The acute treatment with the m-opioid receptor agonist morphine (30 mg/kg, 2 h), compared with saline solution administration, induced a marked increase in the immunodensity of phosphorylated NF-H proteins in the cerebral cortex (93%, n 4, P , 0:001) (Figs. 1B and 2). Chronic treatment with morphine (10±100 mg/kg for 5 days) resulted in the induction of tolerance to the acute effect of the opiate on the phosphorylation of NF-H proteins (Figs. 1B and 2). Moreover, in these morphine-dependent rats, spontaneous opiate withdrawal resulted in a time-dependent (2±24 h) decline in the immunodensity of phosphorylated NF-H proteins. Thus, 2 h after the last dose of morphine (chronic treatment), the immunodensity of phosphorylated NF-H was signi®cantly increased (27%, n 3, P , 0:05), which was followed (6±12 h) by a progressive down-regulation of the phosphorylated protein (5% increase at 6 h, n 3, P . 0:05; 5% decrease at 12 h, n 5, P . 0:05) (Figs. 1B and 2). A spontaneous morphine withdrawal of 24 h was associated with a tendency to decrease the phosphorylation state of NF-H in the brain (29% decrease, n 2; P . 0:05) (Figs. 1B and 2). However, a heroin withdrawal of 24 h in chronic heroin-treated rats (10±30 mg/kg for 5 days) was not associated with signi®cant changes in phos-
P.E. Jaquet et al. / Neuroscience Letters 304 (2001) 37±40
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phorylated NF-H proteins (antibody NE-14) in the brain (% increase in immunoreactivity: 7 ^ 9%, n 3, P . 0:05). Together these results indicate that the chronic effects of morphine and heroin on brain NF-H phosphorylation are not longer evident 12±24 h after the last dose of the opiate. In morphine-treated rats, similar results were obtained when the phosphorylated NF-H proteins in the same brains were immunodetected with the antibody SMI-31 (data not shown). As expected [3], chronic treatment with morphine (10±100 mg/kg for 5 days), but not the acute treatment (30 mg/kg for 2 h), induced a marked decrease in the immunodensity of NF-L proteins in the rat cerebral cortex (86% decrease, n 3, P , 0:01), 2±6 h after the last injection (Fig. 1D). These results con®rmed that the down-regulation of the abundance of NF-L induced by chronic morphine is a speci®c neurochemical marker of opioid addiction. In contrast to morphine, the acute (10 mg/kg for 2 h) and chronic (2 £ 10 mg/kg for 14 days) treatments with naloxone, a non-selective opioid receptor antagonist, did not alter signi®cantly the immunodensity of phosphorylated NF-H proteins in the rat cerebral cortex, although receptor blockade resulted in a clear tendency to decrease the state of NFH phosphorylation (acute treatment: 21 ^ 7% decrease, n 6, P . 0:05; chronic treatment: 16 ^ 10% decrease, n 6, P . 0:05) (Fig. 1C). These results suggest that the opioid receptors (probably the m-type) that modulate the phosphorylation state of NF-H proteins in brain are not tonically activated by endogenous opioid peptides (i.e. endomorphines). The results indicate that both the acute and chronic morphine treatments are associated with increases in the Fig. 1. (A) Effect of dephosphorylation on neuro®lament (NF)-H immunoreactivity for NE-14 antibody (Sigma) in the rat brain (detection of phosphorylated NF-H). Dephosphorylation experiments were performed as described previously [6]. Brain tissue (cerebral cortex) was homogenized, dialyzed, and incubated (duplicate samples) at 308C for 15 min in the absence (control samples) or presence of alkaline phosphatase (75 U, Type XXXT, Sigma). Samples containing the enzyme were also incubated with 100 mM sodium pirophosphate (inhibited controls). (B) Representative immunoblots (same gel) for the effects of saline (S), acute morphine (A, 30 mg/kg, i.p., for 2 h) and chronic morphine (C, 10±100 mg/kg, i.p., for 5 days, and 2, 6, 12 or 24 h after the last dose) treatments on phosphorylated NF-H protein immunoreactivity (antibody NE-14) in the rat cerebral cortex. (C) Representative immunoblots (same gel) for the effects of saline (S), acute naloxone (A, 10 mg/kg, i.p., for 2 h) and chronic naloxone (C, 10 mg/kg, i.p., for 14 days, and 2 h after the last dose) treatments on phosphorylated NF-H protein immunoreactivity (antibody NE-14) in the rat cerebral cortex. (D) Representative immunoblots (same gel) for the effects of saline (S), acute morphine (A, 30 mg/kg, i.p., for 2 h) and chronic morphine (C, 10±100 mg/kg, i.p., for 5 days, and 2 h after the last dose) treatments on nonphosphorylated NF-L protein immunoreactivity (antibody NR-4) in the rat cerebral cortex. The amount of protein loaded on the different gels was 40 mg in all cases. The apparent molecular masses of NF proteins (200 kDa for NF-H and 68 kDa for NF-L) were determined by calibrating the blots with prestained molecular weight markers as shown on the left hand side.
Fig. 2. Effects of acute and chronic morphine treatments on the immunodensity of phosphorylated (antibody NE-14) neuro®lament (pNF)-H proteins in the rat cerebral cortex. Saline (n 12), acute morphine (A, 30 mg/kg, i.p., for 2 h, n 4) and chronic morphine (10±100 mg/kg, i.p., for 5 days, and 2, 6, 12 or 24 h after the last dose, n 3±5, except for 24 h, n 2). Data (bars) are expressed as the mean ^ SEM percentage differences of the saline-treated group. One-way ANOVA (F 5; 23 10:33, P , 0:0001) followed by Bonferroni's multiple comparison test detected signi®cant increases in pNF-H after morphine treatment (*P , 0:05; **P , 0:001).
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amount of phosphorylated NF-H proteins in the rat frontal cortex (revealed with two different phospho-speci®c antibodies), but the acute effect of the opiate is stronger than the chronic effect (induction of tolerance). These results in the rat brain suggest that the observed increases in phosphorylated NF proteins in postmortem brains of opioid addicts [6] can be related to both the chronic effect of the opioid and the ®nal acute effect of the deadly opioid overdose (mainly heroin). This hyperphosphorylation of NF-H proteins might have important consequences in human opioid addiction. For instance, aberrant phosphorylation in NF-H may cause NF proteins to accumulate in the perikaryon and to reduce the rate of axonal transport as well as to alter neuronal morphology [9,12,13]. In fact, direct evidence for impairment of axonal transport following chronic morphine treatment has been demonstrated in rats [2]. Moreover, chronic morphine treatment also has been shown to alter the structure of neurones in brain [15,18]. The mechanism by which morphine alters the size and dendritic structure of neurones is not known, but it may be related to the ability of opiate drugs to alter NF abundance and the state of phosphorylation of these cytoskeletal proteins [1,3,6,15]. This study was funded by Grant 32±57066.99 (Fonds National Suisse de la Recherche Scienti®que), Bern, Switzerland, and by Grant BFI2000±0306 (Fondo Nacional para el Desarrollo de la InvestigacioÂn Cientõ®ca y TeÂcnica), Madrid, Spain. M.F.-A. was supported by a predoctoral fellowship from the FNSRS. J.A.G.-S. is a member of the Institut d'Estudis Catalans (Barcelona, Spain). [1] Beitner-Johnson, D., Guitart, X. and Nestler, E.J., Neuro®lament proteins and the mesolimbic dopamine system: common regulation by chronic morphine and chronic cocaine in the rat ventral tegmental area, J. Neurosci., 12 (1992) 2165±2176. [2] Beitner-Johnson, D. and Nestler, E.J., Chronic morphine impairs mesolimbic transport in the rat mesolimbic dopamine system, NeuroReport, 5 (1993) 57±60. [3] Boronat, M.A., Olmos, G. and GarcõÂa-Sevilla, J.A., Attenuation of tolerance to opioid-induced antinociception and protection against morphine-induced decrease of neuro®lament proteins by idazoxan and other I2-imidazoline ligands, Br. J. Pharmacol., 125 (1998) 175±185. [4] Bradford, M., A rapid and sensitive method for the quantitation of microgram quantities of protein utilising the principle of protein dye-binding, Anal. Biochem., 72 (1976) 248± 252. [5] EscribaÂ, P.V., Sastre, M. and GarcõÂa-Sevilla, J.A., Increased
[6]
[7]
[8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18]
[19]
[20]
density of guanine nucleotide-binding proteins in the postmortem brains of heroin addicts, Arch. Gen. Psychiatry, 51 (1994) 494±501. Ferrer-AlcoÂn, M., GarcõÂa-Sevilla, J.A., Jaquet, P.E., La Harpe, R., Riederer, B.M., Walzer, C. and GuimoÂn, J., Regulation of nonphosphorylated and phosphorylated forms of neuro®lament proteins in the prefrontal cortex of human opioid addicts, J. Neurosci. Res., 61 (2000) 338±349. GarcõÂa-Sevilla, J.A., Ventayol, P., Busquets, X., La Harpe, R., Walzer, C. and GuimoÂn, J., Marked decrease of immunolabelled 68 kDa neuro®lament (NF-L) proteins in brains of opiate addicts, NeuroReport, 8 (1997) 1561±1565. Harlow, E. and Lane, D., Using Antibodies. A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y, 1999. Hirokawa, N. and Takeda, S., Gene targeting studies begin to reveal the function of neuro®lament proteins, J. Cell Biol., 143 (1998) 1±4. Lee, M.K. and Cleveland, D.W., Neuronal intermediate ®laments, Annu. Rev. Neurosci., 19 (1996) 187±217. Nestler, E.J., Berhow, M.T. and Brodkin, E.S., Molecular mechanism of drug addiction: adaptations in signal transduction pathways, Mol. Psychiatry, 1 (1996) 190±199. Nixon, R.A. and Sihag, R.K., Neuro®lament phosphorylation: a new look at regulation and function, Trends Neurosci., 14 (1991) 501±506. Pant, H.C. and Veeranna, Q., Neuro®lament phosphorylation, Biochem. Cell Biol., 73 (1995) 575±592. Robinson, P.A. and Anderton, B.H., Neuro®lament probes: a review of neuro®lament distribution and biology, Rev. Neurosci., 2 (1988) 1±40. Robinson, T.E. and Kolb, B., Morphine alters the structure of neurons in the nucleus accumbens and neocortex of rats, Synapse, 33 (1999) 160±162. Self, D.W. and Nestler, E.J., Molecular mechanisms of drug reinforcement and addiction, Annu. Rev. Neurosci., 18 (1995) 463±495. Simonato, M., The neurochemistry of morphine addiction in the neocortex, Trends Pharmacol. Sci., 17 (1996) 410± 415. Sklair-Tavron, L., Shi, W-X., Lane, S.B., Harris, H.W., Bunney, B.S. and Nestler, E.J., Chronic morphine induces visible changes in the morphology of mesolimbic dopamine neurons, Proc. Natl. Acad. Sci. USA, 93 (1996) 11202±11207. Veeranna, Q., Amin, N.D., Ahn, N.G., Jaffe, H., Winters, C.A., Grant, P. and Pant, H.C., Mitogen-activated protein kinases (Erk1,2) phosphorylate Lys-Ser-Pro (KSP) repeats in neuro®lament proteins NF-H and NF-M, J. Neurosci., 18 (1998) 4008±4021. Ventayol, P., Busquets, X. and GarcõÂa-Sevilla, J.A., Modulation of immunoreactive protein kinase C-a and b isoforms and G proteins by acute and chronic treatments with morphine and other opiate drugs in rat brain, NaunynSchmiedeberg's Arch. Pharmacol., 355 (1997) 491±500.
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Acute and chronic effects of morphine and naloxone on the phosphorylation of neuro®lament-H proteins in the rat brain Philippe E. Jaquet a, Marcel Ferrer-AlcoÂn a, Pedro Ventayol b, Jose GuimoÂn a, JesuÂs A. GarcõÂa-Sevilla a,b,* a
Clinical Research Unit, Department of Psychiatry, University of Geneva, HUG Belle-IdeÂe (Le SaleÁve), 2 Chemin du Petit-Bel-Air, CH-1225 CheÃne-Bourg, Switzerland b Laboratory of Neuropharmacology, Associate Unit of the Institute Cajal/ CSIC, Department of Biology, University of the Balearic Islands, Cra. Valldemossa Km 7.5, E-07071 Palma de Mallorca, Spain Received 30 January 2001; received in revised form 14 March 2001; accepted 14 March 2001
Abstract Increased amounts of phosphorylated neuro®laments (pNF-H and pNF-M) are found in postmortem brains of opioid addicts. Because of the potential relevance of aberrant pNF in opioid addiction (alterations of neuronal cytoskeleton and associated functions), the effects of opiate drugs on pNF-H were investigated in rat brain. Acute morphine (30 mg/kg, 2 h) induced a marked increase in the immunodensity of pNF-H in the cerebral cortex (93%). Chronic morphine (10±100 mg/kg for 5 days) followed by opiate withdrawal resulted in a time-dependent decline in pNF-H (induction of tolerance). Thus, 2 h after the last dose of morphine, the abundance of pNF-H was still increased (27%), which was followed (6±24 h) by down-regulation of pNF-H (5% increase at 6 h; 5% decrease at 12 h, and 29% decrease at 24 h). The acute (10 mg/kg for 2 h) and chronic (2 £ 10 mg/kg for 14 days) treatments with naloxone, an opioid receptor antagonist, did not alter pNF-H in the cerebral cortex, suggesting that the opioid receptors (probably the m-type) modulating the phosphorylation state of NF-H are not tonically activated by endogenous opioids. The results indicate that morphine addiction is associated with an aberrant hyperphophorylation of NF-H in the rat brain. q 2001 Published by Elsevier Science Ireland Ltd. Keywords: Morphine; Naloxone; Opioid addiction; Neuro®lament-H; Phosphorylation; Rat brain
The neuro®lament (NF-H, NF-M and NF-L) proteins (neuronal intermediate ®laments) are the major elements of the cytoskeleton in mature neurones and abundant throughout the perikarya, axons and dendrites [10,14]. Phosphorylation of NF appears to be a major mechanism for NF assembly/disassembly, a process which is essential for the integrity of the neuronal cytoskeleton and associated functions (e.g. axonal transport, axonal plasticity and neuronal morphology [10,12,13,19]. Chronic treatment of rats with morphine has been associated with decreases in the immunodensities of nonphosphorylated NF (mainly NF-L) proteins in the ventral tegmental area [1] and in the frontal cortex [3], two brain regions which are targets for the chronic effects of opioid drugs [5,16,17]. Recently, the immunodensities of nonphosphorylated NF-H and NF-L proteins also were shown to be * Corresponding author. Tel.: 141-22-3055756; fax: 141-223055799. E-mail address: [email protected] (J.A. GarcõÂaSevilla).
decreased in postmortem brains (prefrontal cortex) of chronic opioid abusers [6,7]. In marked contrast, increased immunodensities of phosphorylated NF-H and NF-M were found in the same brains of opioid addicts [6]. These reductions in total NF proteins and the aberrant hyperphosphorylation of NF-H in brains of opioid addicts could play a major role in the cellular mechanisms of opioid addiction [6,11,16]. The increases in phosphorylated NF proteins in brains of opioid addicts [6] could be related to both the chronic effect of the opioid and the ®nal acute effect of the deadly opioid overdose (mainly heroin). Because of the potential relevance of NF phosphorylation in human opioid addiction, this study was designed to assess the in¯uence of the opioid system (acute and chronic effects of morphine and naloxone) on the phosphorylation of NF-H in the rat brain. Male Sprague±Dawley rats (250±300 g) were used. The animals were housed under controlled environmental conditions (228C, 70% humidity, and 12-h light/dark cycle) with free access to food and water. For the acute treatments, the
0304-3940/01/$ - see front matter q 2001 Published by Elsevier Science Ireland Ltd. PII: S03 04 - 394 0( 0 1) 01 72 9- 3
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P.E. Jaquet et al. / Neuroscience Letters 304 (2001) 37±40
rats received a single intraperitoneal (i.p.) injection of morphine (30 mg/kg i.p. for 2 h) or naloxone (10 mg/kg i.p. for 2 h). For the chronic treatment with morphine, the rats were injected i.p. three times daily during 5 days with increasing doses of the opiate (10±100 mg/kg) [5,20], and the animals were sacri®ced 2, 6, 12 or 24 h after the last dose. In another series of experiments, rats were treated with heroin (10±30 mg/kg for 5 days) [20] and the animals were killed 24 h after the last dose. For the chronic treatment with naloxone, the rats received 10 mg/kg i.p. every 12 h during 14 days, and the animals were sacri®ced 2 h after the last dose. Control rats received 0.9% saline vehicle i.p. (1 ml/kg) at the indicated treatment times. The rats were killed by decapitation and specimens of cerebral cortex were dissected and stored at 2808C until use. Morphine HCl and heroin HCl were from UnioÂn-QuõÂmico-FarmaceÂutica S.A.E., Madrid, Spain; and naloxone HCl was from Endo Laboratories, New York, USA. These experiments in rats were performed according to the guidelines of the University of the Balearic Islands, Spain. Preparation of cerebral membranes (containing the cytoskeleton) and quantitation of NF proteins (Western blotting) were assessed as described previously [6,20]. About 100 mg of cerebral frontal cortex was homogenized (1:20, w/v) in 40 mM Tris buffer (pH 7.5) containing 1% Triton X-100, 1 mM EDTA, 1 mM MgCl2, 5 mM NaCl, and the protease inhibitors phenylmethylsulfonyl ¯uoride (1 mM) and leupeptin (40 mg/ml). The samples were centrifuged at 40 000 £ g for 45 min, and then 100 ml of the resulting supernatant (containing the cytoskeleton) was mixed with an equal volume of electrophoresis loading buffer [20], which was then boiled (denatured) and stored at 2208C until use. Proteins were determined by the method of Bradford [4]. 40 mg protein of each brain sample was submitted to SDS-PAGE on 15-well (6 £ 8 cm minigels, 1.0 mm thick) 7% polyacrylamide gels. Proteins were electrophoretically transferred to nitrocellulose membranes (immunoblotting) that were incubated (1 h) in phosphate-buffered saline containing 5% nonfat dry milk, 0.2% Tween 20 and 0.5% bovine serum albumine (blocking solution) [8]. Then the membranes were incubated overnight at 48C in blocking solution containing the appropriate primary monoclonal antibody: phosphorylated NF-H (anti-NF 200 kDa, clone NE-14, Lot 056H4812, 1:1000 dilution, Sigma BioSciences, St. Louis, MO, USA; and antibody SMI-31, 1:5000 dilution, batch 13, Sternberger Monoclonals, Baltimore, MD, USA) and nonphosphorylated NF-L (anti-NF 68 kDa, clone NR-4, Lot 083H4811, 1:500 dilution, Sigma BioSciences). The secondary antibody, sheep anti-mouse IgG was incubated at 1:7,000 dilution in blocking solution for 2 h. Immunoreactivity was detected with an enhanced chemoluminescence (ECL) western blot detection system (Amersham, Buckinghamshire, UK), followed by exposure to Amersham Hyper®lm ECL for 1±10 min. The autoradiograms were quantitated by densitometric scanning (GS-700 Imaging Densitometer, Bio-Rad, Hercules, CA, USA) by measuring
the integrated optical density (IOD) of the immunoreactive bands. The amount of NF proteins in the prefrontal cortex of rats treated with opiates was compared in the same gel with that of control rats, which received saline solution. Prior to analyses, the linearity of protein concentration for Western blotting was ascertained by resolution of selected concentrations of protein (i.e., total protein loaded versus IOD units, consisting of six points of different protein content, 10 to 60 mg, resulting in linear relations) (data not shown). Experiments were performed by using 40 mg known to be within the linear range for immunolabeling of the target proteins. This quanti®cation procedure was assessed twice in different gels. Finally, percent changes in immunoreactivity with respect to control samples (100%) were calculated for each rat treated with morphine, heroin or naloxone in the two gels and the mean value used as a ®nal estimate. Results are expressed as the mean ^ SEM values. One-way analysis of variance (ANOVA), followed by Bonferroni's multiple comparison test, was used for the statistical evaluation. The level of signi®cance was chosen as P 0:05: Immunoreactive bands of 200 kDa and 68 kDa, corresponding respectively to the NF-H and NF-L cytoskeletal proteins [6,10,14], were found in the frontal cortex of rats after immunoblotting (Fig. 1). In preliminary experiments NF antibodies were tested for their speci®city for phosphorylated NF-H proteins. Antibodies NE-14 (Fig. 1A) and SMI-31 (data not shown) (see Ref. [6]) reacted speci®cally with phosphorylated epitopes of NF-H, because its enzymatic dephosphorylation with alkaline phosphatase completely abolished the immunoreactivity of the antibodies (see Fig. 1A). The immunoreactivity of phospho-speci®c antibodies against NF-H re¯ects the phosphorylationdependent alteration of NF organization [12,13]. The acute treatment with the m-opioid receptor agonist morphine (30 mg/kg, 2 h), compared with saline solution administration, induced a marked increase in the immunodensity of phosphorylated NF-H proteins in the cerebral cortex (93%, n 4, P , 0:001) (Figs. 1B and 2). Chronic treatment with morphine (10±100 mg/kg for 5 days) resulted in the induction of tolerance to the acute effect of the opiate on the phosphorylation of NF-H proteins (Figs. 1B and 2). Moreover, in these morphine-dependent rats, spontaneous opiate withdrawal resulted in a time-dependent (2±24 h) decline in the immunodensity of phosphorylated NF-H proteins. Thus, 2 h after the last dose of morphine (chronic treatment), the immunodensity of phosphorylated NF-H was signi®cantly increased (27%, n 3, P , 0:05), which was followed (6±12 h) by a progressive down-regulation of the phosphorylated protein (5% increase at 6 h, n 3, P . 0:05; 5% decrease at 12 h, n 5, P . 0:05) (Figs. 1B and 2). A spontaneous morphine withdrawal of 24 h was associated with a tendency to decrease the phosphorylation state of NF-H in the brain (29% decrease, n 2; P . 0:05) (Figs. 1B and 2). However, a heroin withdrawal of 24 h in chronic heroin-treated rats (10±30 mg/kg for 5 days) was not associated with signi®cant changes in phos-
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phorylated NF-H proteins (antibody NE-14) in the brain (% increase in immunoreactivity: 7 ^ 9%, n 3, P . 0:05). Together these results indicate that the chronic effects of morphine and heroin on brain NF-H phosphorylation are not longer evident 12±24 h after the last dose of the opiate. In morphine-treated rats, similar results were obtained when the phosphorylated NF-H proteins in the same brains were immunodetected with the antibody SMI-31 (data not shown). As expected [3], chronic treatment with morphine (10±100 mg/kg for 5 days), but not the acute treatment (30 mg/kg for 2 h), induced a marked decrease in the immunodensity of NF-L proteins in the rat cerebral cortex (86% decrease, n 3, P , 0:01), 2±6 h after the last injection (Fig. 1D). These results con®rmed that the down-regulation of the abundance of NF-L induced by chronic morphine is a speci®c neurochemical marker of opioid addiction. In contrast to morphine, the acute (10 mg/kg for 2 h) and chronic (2 £ 10 mg/kg for 14 days) treatments with naloxone, a non-selective opioid receptor antagonist, did not alter signi®cantly the immunodensity of phosphorylated NF-H proteins in the rat cerebral cortex, although receptor blockade resulted in a clear tendency to decrease the state of NFH phosphorylation (acute treatment: 21 ^ 7% decrease, n 6, P . 0:05; chronic treatment: 16 ^ 10% decrease, n 6, P . 0:05) (Fig. 1C). These results suggest that the opioid receptors (probably the m-type) that modulate the phosphorylation state of NF-H proteins in brain are not tonically activated by endogenous opioid peptides (i.e. endomorphines). The results indicate that both the acute and chronic morphine treatments are associated with increases in the Fig. 1. (A) Effect of dephosphorylation on neuro®lament (NF)-H immunoreactivity for NE-14 antibody (Sigma) in the rat brain (detection of phosphorylated NF-H). Dephosphorylation experiments were performed as described previously [6]. Brain tissue (cerebral cortex) was homogenized, dialyzed, and incubated (duplicate samples) at 308C for 15 min in the absence (control samples) or presence of alkaline phosphatase (75 U, Type XXXT, Sigma). Samples containing the enzyme were also incubated with 100 mM sodium pirophosphate (inhibited controls). (B) Representative immunoblots (same gel) for the effects of saline (S), acute morphine (A, 30 mg/kg, i.p., for 2 h) and chronic morphine (C, 10±100 mg/kg, i.p., for 5 days, and 2, 6, 12 or 24 h after the last dose) treatments on phosphorylated NF-H protein immunoreactivity (antibody NE-14) in the rat cerebral cortex. (C) Representative immunoblots (same gel) for the effects of saline (S), acute naloxone (A, 10 mg/kg, i.p., for 2 h) and chronic naloxone (C, 10 mg/kg, i.p., for 14 days, and 2 h after the last dose) treatments on phosphorylated NF-H protein immunoreactivity (antibody NE-14) in the rat cerebral cortex. (D) Representative immunoblots (same gel) for the effects of saline (S), acute morphine (A, 30 mg/kg, i.p., for 2 h) and chronic morphine (C, 10±100 mg/kg, i.p., for 5 days, and 2 h after the last dose) treatments on nonphosphorylated NF-L protein immunoreactivity (antibody NR-4) in the rat cerebral cortex. The amount of protein loaded on the different gels was 40 mg in all cases. The apparent molecular masses of NF proteins (200 kDa for NF-H and 68 kDa for NF-L) were determined by calibrating the blots with prestained molecular weight markers as shown on the left hand side.
Fig. 2. Effects of acute and chronic morphine treatments on the immunodensity of phosphorylated (antibody NE-14) neuro®lament (pNF)-H proteins in the rat cerebral cortex. Saline (n 12), acute morphine (A, 30 mg/kg, i.p., for 2 h, n 4) and chronic morphine (10±100 mg/kg, i.p., for 5 days, and 2, 6, 12 or 24 h after the last dose, n 3±5, except for 24 h, n 2). Data (bars) are expressed as the mean ^ SEM percentage differences of the saline-treated group. One-way ANOVA (F 5; 23 10:33, P , 0:0001) followed by Bonferroni's multiple comparison test detected signi®cant increases in pNF-H after morphine treatment (*P , 0:05; **P , 0:001).
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amount of phosphorylated NF-H proteins in the rat frontal cortex (revealed with two different phospho-speci®c antibodies), but the acute effect of the opiate is stronger than the chronic effect (induction of tolerance). These results in the rat brain suggest that the observed increases in phosphorylated NF proteins in postmortem brains of opioid addicts [6] can be related to both the chronic effect of the opioid and the ®nal acute effect of the deadly opioid overdose (mainly heroin). This hyperphosphorylation of NF-H proteins might have important consequences in human opioid addiction. For instance, aberrant phosphorylation in NF-H may cause NF proteins to accumulate in the perikaryon and to reduce the rate of axonal transport as well as to alter neuronal morphology [9,12,13]. In fact, direct evidence for impairment of axonal transport following chronic morphine treatment has been demonstrated in rats [2]. Moreover, chronic morphine treatment also has been shown to alter the structure of neurones in brain [15,18]. The mechanism by which morphine alters the size and dendritic structure of neurones is not known, but it may be related to the ability of opiate drugs to alter NF abundance and the state of phosphorylation of these cytoskeletal proteins [1,3,6,15]. This study was funded by Grant 32±57066.99 (Fonds National Suisse de la Recherche Scienti®que), Bern, Switzerland, and by Grant BFI2000±0306 (Fondo Nacional para el Desarrollo de la InvestigacioÂn Cientõ®ca y TeÂcnica), Madrid, Spain. M.F.-A. was supported by a predoctoral fellowship from the FNSRS. J.A.G.-S. is a member of the Institut d'Estudis Catalans (Barcelona, Spain). [1] Beitner-Johnson, D., Guitart, X. and Nestler, E.J., Neuro®lament proteins and the mesolimbic dopamine system: common regulation by chronic morphine and chronic cocaine in the rat ventral tegmental area, J. Neurosci., 12 (1992) 2165±2176. [2] Beitner-Johnson, D. and Nestler, E.J., Chronic morphine impairs mesolimbic transport in the rat mesolimbic dopamine system, NeuroReport, 5 (1993) 57±60. [3] Boronat, M.A., Olmos, G. and GarcõÂa-Sevilla, J.A., Attenuation of tolerance to opioid-induced antinociception and protection against morphine-induced decrease of neuro®lament proteins by idazoxan and other I2-imidazoline ligands, Br. J. Pharmacol., 125 (1998) 175±185. [4] Bradford, M., A rapid and sensitive method for the quantitation of microgram quantities of protein utilising the principle of protein dye-binding, Anal. Biochem., 72 (1976) 248± 252. [5] EscribaÂ, P.V., Sastre, M. and GarcõÂa-Sevilla, J.A., Increased
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