sl-327 and Substance-Withdrawal-Syndrome

Epigenetic changes such as covalent modifications of histone proteins represent complex molecular signatures that provide a cellular memory of previously experienced stimuli without irreversible changes of the genetic code. In this study we show that new gene expression induced in vivo by morphine withdrawal occurs with concomitant epigenetic modifications in brain regions critically involved in drug-dependent behaviors. We found that naloxone-precipitated withdrawal, but not chronic morphine administration, caused a strong induction of phospho-histone H3 immunoreactivity in the nucleus accumbens (NAc) shell/core and in the lateral septum (LS), a change that was accompanied by augmented H3 acetylation (lys14) in neurons of the NAc shell. Morphine withdrawal induced the phosphorylation of the epigenetic factor methyl-CpG-binding protein 2 (MeCP2) in Ser421 both in the LS and the NAc shell. These epigenetic changes were accompanied by the activation of members of the ERK pathway as well as increased expression of the immediate early genes (IEG) c-fos and activity-regulated cytoskeleton-associated protein (Arc/Arg3.1). Using a pharmacological approach, we found that H3 phosphorylation and IEG expression were partially dependent on ERK activation, while MeCP2 phosphorylation was fully ERK-independent. These findings provide new important information on the role of the ERK pathway in the regulation of epigenetic marks and gene expression that may concur to regulate in vivo the cellular changes underlying the onset of the opioid withdrawal syndrome.

Topics: Acetylation; Aminoacetonitrile; Animals; Cytoskeletal Proteins; Epigenesis, Genetic; Gene Expression; Histones; Male; MAP Kinase Signaling System; Methyl-CpG-Binding Protein 2; Morphine; Naloxone; Nerve Tissue Proteins; Neurons; Nucleus Accumbens; Phosphorylation; Proto-Oncogene Proteins c-fos; Rats; Septal Nuclei; Signal Transduction; Substance Withdrawal Syndrome

The negative affective states of withdrawal involve the recruitment of brain and peripheral stress circuitry [e.g., noradrenergic activity, induction of the hypothalamo-pituitary-adrenocortical (HPA) axis, and the expression and activation of heat shock proteins (Hsps)]. The present study investigated the role of extracellular signal-regulated protein kinase (ERK) and β-adrenoceptor on the response of stress systems to morphine withdrawal by the administration of [amino[(4-aminophenyl)thio]methylene]-2-(trifluoromethyl)benzeneacetonitrile (SL327), a selective inhibitor of ERK activation, or propranolol (a β-adrenoceptor antagonist). Dependence on morphine was induced by a 7-day subcutaneous implantation of morphine pellets. Morphine withdrawal was precipitated on day 8 by the injection of naloxone (2 mg/kg s.c.). Plasma concentrations of adrenocorticotropin and corticosterone were determined by radioimmunoassay; noradrenaline (NA) turnover in left ventricle was determined by high-performance liquid chromatography; and catechol-O-methyl transferase (COMT) and Hsp27 expression and phosphorylation at Ser82 were determined by quantitative blot immunolabeling. Morphine-withdrawn rats showed an increase of NA turnover and COMT expression in parallel with an enhancement of adrenocorticotropin and plasma corticosterone concentrations. In addition, we observed an enhancement of Hsp27 expression and phosphorylation. Pretreatment with SL327 or propranolol significantly reduced morphine withdrawal-induced increases of plasma adrenocorticotropin and Hsp27 phosphorylation at Ser82 without any changes in plasma corticosterone levels. The present findings demonstrate that morphine withdrawal is capable of inducing the activation of HPA axis in parallel with an enhancement of Hsp27 expression and Hsp27 phosphorylation at Ser82 and suggest a role for β-adrenoceptors and ERK pathways in mediating morphine-withdrawal activation of the HPA axis and cellular stress response.

Topics: Adrenergic beta-Antagonists; Adrenocorticotropic Hormone; Aminoacetonitrile; Animals; Catechol O-Methyltransferase; Corticosterone; Extracellular Signal-Regulated MAP Kinases; Heart; Heart Ventricles; HSP27 Heat-Shock Proteins; Hypothalamo-Hypophyseal System; Male; Morphine; Morphine Dependence; Naloxone; Norepinephrine; Phosphorylation; Pituitary-Adrenal System; Propranolol; Rats; Rats, Sprague-Dawley; Receptors, Adrenergic, beta; Substance Withdrawal Syndrome

Heat shock protein 27 (Hsp27) is a well-known stress response protein that becomes phosphorylated through extracellular signal-regulated kinase (ERK). Different drugs of abuse, such as morphine and/or its withdrawal, induce severe stress situations. In this study, we investigated Hsp27 and phospho-Hsp27 expression during morphine dependence and withdrawal and evaluated the involvement of ERK in the phosphorylation of Hsp27 in the rat right ventricle. Dependence on morphine was induced by a 7-day s.c. implantation of morphine pellets. Morphine withdrawal was precipitated on day 8 by injection of naloxone (2 mg/kg, s.c.). ERK1/2, Hsp27 and phospho-Hsp27 at Ser15 were determined by quantitative blot immunolabeling using specific antibodies. Hsp27 expression was increased 30, 60, 90 and 120 min (144.5±14.2%, P<0.0001; 128.9±4.6%, P=0.04; 177.4±12.7, P<0.0001; and 136.2±11.0%, P=0.042, respectively) after saline injection to rats dependent on morphine. Naloxone-precipitated morphine withdrawal also increased the phosphorylation of Hsp27 at Ser15 at those time points (146.8±19.8%, P=0.034; 143.9±17.9%, P=0.032; 161.2±33.3%, P=0.029; and 152.2±25.5%, P=0.008, respectively). However, there were no changes in Hsp27 phosphorylation in the morphine dependent group injected with saline. In addition, there was an increase in the phosphorylation of ERK 60 min after naloxone injection in morphine dependent rats (pERK1: 116.3±4.2%, P=0.015 and pERK2: 117.2±1.5%, P=0.05). Pretreatment with SL327, an inhibitor of ERK phosphorylation, decreased activation (phosphorylation) of both ERK and Hsp27 (pERK1: 4.5±3.6%, P<0.0001; pERK2: 42.3±3.3%, P<0.0001; and pHsp27: 97.6±1.5%, P=0.008), suggesting that ERK activation triggers Hsp27 phosphorylation. The present findings demonstrate that morphine withdrawal is capable of inducing the activation of Hsp27 in the heart and suggest that phosphorylation of Hsp27 is closely linked to and also dependent on the ERK pathway.

Topics: Aminoacetonitrile; Animals; Extracellular Signal-Regulated MAP Kinases; Heart; Heart Ventricles; HSP27 Heat-Shock Proteins; Immunoblotting; Morphine; Morphine Dependence; Myocardium; Naloxone; Phosphorylation; Protease Inhibitors; Rats; Rats, Sprague-Dawley; Substance Withdrawal Syndrome

Drugs of abuse induce behavioral neuroadaptations whose molecular mechanisms, partly known, are crucial to understanding drug addictions. The multifunctional adaptor Fas-associated protein with death domain (FADD) was recently associated with the induction of neuroplasticity. This study investigated the modulation of FADD and MAP kinase signaling, as well as their interactions with PEA-15 (phosphoprotein enriched in astrocytes-15 kDa) and Akt1 pathways, during the expression of unconditioned morphine-induced psychomotor sensitization. In morphine-pretreated rats (10mg/kg during 5 days), a challenge dose of the opiate induced a robust psychomotor sensitization at early withdrawal (3 days, SW 3), but not after a prolonged abstinence period (14 days), which was coincident with an accelerated dopamine turnover in the striatum. Marked concomitant increases in the content of p-FADD (48%) and the activation of MEK-ERK (46-79%) were quantified during the short-term expression of morphine sensitization (SW 3, in the absence of morphine challenge). At SW 3, p-PEA-15, a FADD-ERK binding partner, was also upregulated (51%) as well as the activation of its phosphorylating Akt1 kinase (49%). Notably, the MEK inhibitor SL 327 attenuated (58%) the expression of morphine-induced psychomotor sensitization (SW 3) and fully prevented the upregulation of p-FADD, p-PEA-15 and p-Akt1 at SW 3. The results indicate that the activation of MEK/ERK, the upregulation of p-FADD and that of the linking partners PEA-15/Akt1 have a major role in mediating the short-lasting expression of unconditioned psychomotor sensitization induced by morphine in rats.

Topics: Aminoacetonitrile; Analysis of Variance; Animals; Apoptosis; Brain Chemistry; Chromatography, High Pressure Liquid; Corpus Striatum; Drug Administration Schedule; Electrochemistry; Enzyme Inhibitors; Extracellular Signal-Regulated MAP Kinases; Fas-Associated Death Domain Protein; Gene Expression Regulation; JNK Mitogen-Activated Protein Kinases; Locomotion; Male; Morphine; Narcotics; Nerve Tissue Proteins; Phosphorylation; Protein Binding; Psychomotor Performance; Rats; Rats, Sprague-Dawley; Signal Transduction; Substance Withdrawal Syndrome; Time Factors

Our previous studies have shown that morphine withdrawal induced an increase in the expression of protein kinase (PK) A and mitogen-activated extracellular kinase (MAPK) pathways in the heart during morphine withdrawal. The purpose of the present study was to evaluate the interaction between PKA and extracellular signal-regulated kinase (ERK) signaling pathways mediating the cardiac adaptive changes observed after naloxone administration to morphine-dependent rats. Dependence on morphine was induced by a 7-day subcutaneous implantation of morphine pellets. Morphine withdrawal was precipitated on day 8 by an injection of naloxone (2 mg/kg). ERK1/2 and tyrosine hydroxylase (TH) phosphorylation was determined by quantitative blot immunolabeling using phosphorylation state-specific antibodies. Naloxone-induced morphine withdrawal activates ERK1/2 and phosphorylates TH at Ser31 in the right and left ventricle, with an increase in the mean arterial blood pressure and heart rate. When N-(2-guanidinoethyl)-5-isoquinolinesulfonamide (HA-1004), a PKA inhibitor, was infused, concomitantly with morphine, it diminished the expression of ERK1/2. In contrast, the infusion of calphostin C (a PKC inhibitor) did not modify the morphine withdrawal-induced activation of ERK1/2. The ability of morphine withdrawal to activate ERK that phosphorylates TH at Ser31 was reduced by HA-1004. The present findings demonstrate that the enhancement of ERK1/2 expression and the phosphorylation state of TH at Ser31 during morphine withdrawal are dependent on PKA and suggest cross-talk between PKA and ERK1/2 transduction pathway mediating morphine withdrawal-induced activation (phosphorylation) of TH.

Topics: Adaptation, Physiological; Aminoacetonitrile; Analgesics, Opioid; Animals; Blotting, Western; Cyclic AMP-Dependent Protein Kinases; Enzyme Inhibitors; Heart; Hemodynamics; Isoquinolines; Male; Mitogen-Activated Protein Kinases; Morphine; Naloxone; Naphthalenes; Narcotic Antagonists; Phosphorylation; Protein Kinase C; Rats; Rats, Sprague-Dawley; Signal Transduction; Substance Withdrawal Syndrome; Sulfonamides; Tyrosine 3-Monooxygenase; Weight Gain

This study examined whether activation of extracellular signal-regulated kinase (ERK) contributes to the increased open-arm time observed in the elevated plus maze (EPM) during opioid withdrawal. We applied SL327, a selective ERK kinase (MEK) inhibitor, to specific limbic areas and examined the effect on EPM behaviors of controls and during naloxone-precipitated morphine withdrawal. We next confirmed that ERK activation increased in limbic areas of mice undergoing naloxone-precipitated morphine withdrawal. Direct injection of SL327 into the amygdala blocked the withdrawal-induced increase in open-arm time; however, injecting SL327 into the septum had no effect. Consistent with these results, both 0.2 and 2 mg/kg naloxone increased ERK activation in the central amygdala of morphine-dependent mice. In drug-naive mice, 2 mg/kg naloxone, but not 0.2 mg/kg, increased ERK activation in the central amygdala. During withdrawal, increased ERK activation was also observed in the lateral septum. In the locus coeruleus, a significant increase was observed only in morphine-dependent mice receiving 2 mg/kg, but not 0.2 mg/kg naloxone. In conclusion, ERK activation in limbic areas is likely involved in both the aversive properties of naloxone and in the affective/emotional symptoms of opioid withdrawal, including mediating EPM behaviors.

Topics: Aminoacetonitrile; Amygdala; Animals; Enzyme Activation; Extracellular Signal-Regulated MAP Kinases; Male; Maze Learning; Mice; Mice, Inbred C57BL; Morphine; Naloxone; Septum of Brain; Substance Withdrawal Syndrome

Our previous studies have shown that morphine withdrawal induced hyperactivity of cardiac noradrenergic pathways. The purpose of the present study was to evaluate the effects of morphine withdrawal on site-specific phosphorylation of TH in the heart.. Dependence on morphine was induced by a 7-day s.c. implantation of morphine pellets in rats. Morphine withdrawal was precipitated on day 8 by an injection of naloxone (2 mg kg(-1)). TH phosphorylation was determined by quantitative blot immunolabelling using phosphorylation state-specific antibodies.. Naloxone-induced morphine withdrawal induced phosphorylation of TH at serine (Ser)40 and Ser31 in the right ventricle, associated with both an increase in total TH levels and an enhancement of TH activity. When HA-1004 (PK A inhibitor) was infused, concomitantly with morphine, it diminished the increase in noradrenaline turnover, total TH levels and TH phosphorylation at Ser40 in morphine-withdrawn rats. In contrast, the infusion of calphostin C (PKC inhibitor), did not modify the morphine withdrawal-induced increase in noradrenaline turnover and total TH levels. In addition, we show that the ability of morphine withdrawal to stimulate phosphorylation at Ser31 was reduced by SL327, an inhibitor of ERK 1/2 activation.. The present findings demonstrate that the enhancement of total TH levels and the increased phosphorylation state of TH during morphine withdrawal were dependent on PKA and ERK activities and suggest that these transduction pathways might contribute to the activation of the cardiac catecholaminergic neurons in response to morphine withdrawal.

Topics: Aminoacetonitrile; Animals; Cyclic AMP-Dependent Protein Kinases; Disease Models, Animal; Drug Implants; Isoquinolines; Male; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Morphine; Morphine Dependence; Myocardium; Naloxone; Naphthalenes; Narcotic Antagonists; Norepinephrine; Phosphorylation; Protein Kinase C-delta; Protein Kinase Inhibitors; Rats; Rats, Sprague-Dawley; Serine; Substance Withdrawal Syndrome; Sulfonamides; Tyrosine 3-Monooxygenase

Our previous studies have shown that morphine withdrawal increases the hypothalamic-pituitary-adrenocortical axis activity, which is dependent on a hyperactivity of noradrenergic pathways (nucleus tractus solitarius-A(2)) innervating the hypothalamic paraventricular nucleus. The extracellular signal-regulated kinase has been implicated in drug addiction, but its role in activation of paraventricular nucleus and nucleus tractus solitarius during morphine dependence remain poorly understood. We have determined the activation of extracellular signal-regulated kinase during morphine dependence and withdrawal as well as its involvement in morphine withdrawal-induced gene expression. We show that naloxone-induced morphine withdrawal activates extracellular signal-regulated kinases(1/2) and increases c-Fos expression in rat paraventricular nucleus and nucleus tractus solitarius-A(2) neurons. Activated extracellular signal-regulated kinases(1/2) was colocalized with c-Fos in both nuclei, and this response was blocked by SL327, a drug that prevents extracellular signal-regulated kinase activation. In the paraventricular nucleus from morphine-withdrawn rats, the number of neurons expressing CRF was increased. Immunohistochemical study showed a dramatic increase in c-Fos immunoreactivity within CRF-positive cells. These results suggest that extracellular signal-regulated kinases1/2 signaling pathway is necessary for morphine withdrawal-induced activation of brain areas associated with the stress system.

Topics: Aminoacetonitrile; Animals; Blotting, Western; Brain; Corticotropin-Releasing Hormone; Extracellular Signal-Regulated MAP Kinases; Immunochemistry; Injections, Subcutaneous; Male; Morphine; Morphine Dependence; Naloxone; Narcotic Antagonists; Narcotics; Paraventricular Hypothalamic Nucleus; Protease Inhibitors; Proto-Oncogene Proteins c-fos; Rats; Rats, Sprague-Dawley; Signal Transduction; Solitary Nucleus; Substance Withdrawal Syndrome; Time Factors

We have previously demonstrated that morphine withdrawal induced hyperactivity of the heart by activation of noradrenergic pathways innervating the left and right ventricle, as evaluated by noradrenaline turnover and c-Fos expression. The extracellular signal-regulated kinase (ERK) has been implicated in drug addiction, but its role in activation of the heart during morphine dependence remains poorly understood. Here, we have looked for activation of ERK during morphine withdrawal and if this activation induced gene expression.. Dependence on morphine was induced by s.c. implantation of morphine pellets for 7 days. Morphine withdrawal was precipitated on day 8 by injection of naloxone (2 mg kg(-1), s.c.). ERK1/2, their phosphorylated forms and c-Fos were measured by western blotting and immunohistochemistry of cardiac tissue.. Naloxone-induced morphine withdrawal activated ERK1/2 and increased c-Fos expression in cardiac tissues. c-Fos expression was blocked by SL327, a drug that prevents ERK activation.. These results indicate that signalling through the ERKs is necessary for morphine withdrawal-induced hyperactivity of the heart and suggest that this pathway may also be involved in activation of immediate-early genes in both cytosolic and nuclear effector mechanisms that have the potential to bring about long-term changes in the heart.

Topics: Aminoacetonitrile; Animals; Blotting, Western; Extracellular Signal-Regulated MAP Kinases; Gene Expression; Genes, fos; Heart Ventricles; Immunohistochemistry; Male; MAP Kinase Signaling System; Morphine; Naloxone; Narcotic Antagonists; Narcotics; Phosphorylation; Rats; Rats, Sprague-Dawley; Signal Transduction; Substance Withdrawal Syndrome

The clinical use of opioids is limited by the development of tolerance and physical dependence. Opioid tolerance and dependence are believed to result from complex adaptations in the CNS, representing a form of neural plasticity. Extracellular signal-regulated kinases (ERKs) are involved in many forms of neural plasticity, and therefore could also be involved in the development of opioid tolerance and dependence. In this study, we investigated the effect of a systemically bioavailable MEK (ERK kinase) inhibitor, SL327, upon the development and the expression of tolerance to and dependence on morphine in mice. In tolerance and dependence development studies, two strains of mice were treated daily for 8 or 9 days with 5mg/kg morphine s.c. Tolerance development was assessed by tail flick latency. Withdrawal was then precipitated by subcutaneous injection of 2mg/kg naloxone s.c. and signs recorded. Co-administration of 50mg/kg SL327 i.p. prior to morphine administration had no effect on the development of tolerance or withdrawal signs. To study possible effects of ERK inhibition on the expression of tolerance and dependence, mice were implanted with 75mg morphine pellets s.c. Tolerance and dependence were assessed as previously described. An acute i.p. injection of 50mg/kg SL327 after 4 days of morphine exposure had no effect on the expression of either morphine tolerance or physical dependence. To verify that this dose of SL327 inhibited morphine-induced ERK modulation, mice received an acute i.p. injection of 50mg/kg SL327 prior to morphine administration, and sacrificed 30min later. Western blots demonstrated that SL327 did inhibit morphine-induced ERK modulation. Taken together, these data suggest that unlike many other observed forms of neural plasticity, the ERK signaling cascade is not involved in the development or expression of opioid tolerance and dependence.

Topics: Aminoacetonitrile; Animals; Blotting, Western; Dose-Response Relationship, Drug; Drug Tolerance; Enzyme Inhibitors; Extracellular Signal-Regulated MAP Kinases; Male; Mice; Mice, Inbred C57BL; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Morphine Dependence; Naloxone; Narcotic Antagonists; Neuronal Plasticity; Pain Measurement; Reaction Time; Substance Withdrawal Syndrome