Proteomic Data in Morphine Addiction Versus Real Protein Activity: Metabolic Enzymes.

Drug dependence is an escalating problem worldwide and many efforts are being made to understand the molecular basis of addiction. The morphine model is widely used in these investigations. To date, at least 29 studies exploring the influence of morphine on mammals' proteomes have been published. Among various proteins indicated as up- or down-regulated, the expression changes of enzymes engaged in energy metabolism pathways have often been confirmed. To verify whether proteomics-indicated alterations in enzyme levels reflect changes in their activity, four enzymes: PK, MDH, Complex I, and Complex V were investigated in morphine addiction and abstinence models. After analyses of the rat brain mitochondria fraction in the model of morphine dependence, we found that one of the investigated enzymes (pyruvate kinase) showed statistically significant differences observed between morphine, control, and abstinence groups. J. Cell. Biochem. 118: 4323-4330, 2017. © 2017 Wiley Periodicals, Inc.

Loss of morphine reward and dependence in mice lacking G protein-coupled receptor kinase 5.

BACKGROUND: The clinical benefits of opioid drugs are counteracted by the development of tolerance and addiction. We provide in vivo evidence for the involvement of G protein-coupled receptor kinases (GRKs) in opioid dependence in addition to their roles in agonist-selective mu-opioid receptor (MOR) phosphorylation. METHODS: In vivo MOR phosphorylation was examined by immunoprecipitation and nanoflow liquid chromatography-tandem mass spectrometry analysis. Using the hot-plate and conditioned place preference test, we investigated opioid-related antinociception and reward effects in mice lacking GRK3 or GRK5. RESULTS: Etonitazene and fentanyl stimulated the in vivo phosphorylation of multiple carboxyl-terminal phosphate acceptor sites, including threonine 370, serine 375, and threonine 379, which was predominantly mediated by GRK3. By contrast, morphine promoted a selective phosphorylation of serine 375 that was predominantly mediated by GRK5. In contrast to GRK3 knockout mice, GRK5 knockout mice exhibited reduced antinociceptive responses after morphine administration and developed morphine tolerance similar to wild-type mice but fewer signs of physical dependence. Also, morphine was ineffective in inducing conditioned place preference in GRK5 knockout mice, whereas cocaine conditioned place preference was retained. However, the reward properties of morphine were evident in knock-in mice expressing a phosphorylation-deficient S375A mutation of the MOR. CONCLUSIONS: These findings show for the first time that MOR phosphorylation is regulated by agonist-selective recruitment of distinct GRK isoforms that influence different opioid-related behaviors. Modulation of GRK5 function could serve as a new approach for preventing addiction to opioids, while maintaining the analgesic properties of opioid drugs at an effective level.

Pharmacological modulation of geranylgeranyltransferase and farnesyltransferase attenuates opioid withdrawal in vivo and in vitro.

Geranylgeranyltransferase and farnesyltransferase I, are noted to mediate a number of signal transduction cascades which are known to be involved in the causation of opioid withdrawal syndrome. GGTI-2133 and FTI-276 are selective modulators of geranylgeranyltransferase and farnesyltransferase subtype 1 respectively. Therefore, the present study investigated the effect of GGTI-2133 and FTI-276 on propagation of morphine dependence and resultant withdrawal signs in vivo, in sub-chronic morphine mouse model, and in vitro, in isolated rat ileum. Morphine was administered twice daily for 5 days following which a single day 6 injection of naloxone (8 mg/kg, i.p.) precipitated opioid withdrawal syndrome in mice. Withdrawal syndrome was quantitatively assessed in terms of withdrawal severity score and the frequency of jumping, rearing, fore paw licking & circling. Naloxone induced contraction in morphine withdrawn isolated rat ileum was employed as an in vitro model of opioid withdrawal syndrome. An isobolographic study design was employed to assess a potential synergistic activity between GGTI-2133 and FTI-276. GGTI-2133 and FTI-276 dose dependently attenuated naloxone induced morphine withdrawal syndrome both in vivo and in vitro. GGTI-2133 was also observed to exert a synergistic interaction with FTI-276. It is concluded that GGTI-2133 and FTI-276 attenuate the propagation of morphine dependence and reduce withdrawal signs possibly by a geranylgeranyl transferase; farnesyltransferase activation pathway linked mechanisms potentially in an interdependent manner.

Dual inhibition of endocannabinoid catabolic enzymes produces enhanced antiwithdrawal effects in morphine-dependent mice.

Inhibition of the endocannabinoid catabolic enzymes, monoacylglycerol lipase (MAGL) or fatty acid amide hydrolase (FAAH) attenuates naloxone-precipitated opioid withdrawal signs in mice via activation of CB1 receptors. Complete FAAH inhibition blocks only a subset of withdrawal signs, whereas complete MAGL inhibition elicits enhanced antiwithdrawal efficacy, but is accompanied with some cannabimimetic side effects. Thus, the primary objective of the present study was to determine whether combined, full FAAH inhibition and partial MAGL represents an optimal strategy to reduce opioid withdrawal. To test this hypothesis, we examined whether combined administration of high-dose of the FAAH inhibitor PF-3845 and low-dose of the MAGL inhibitor JZL184, as well as the novel dual FAAH-MAGL inhibitor SA-57, which is 100-fold more potent in inhibiting FAAH than MAGL, would prevent spontaneous withdrawal in morphine-dependent mice, a model with greater face validity than precipitating withdrawal with µ-opioid receptor antagonists. Strikingly, a combination of low-dose JZL184 and high-dose PF-3845 as well as the dual inhibitor SA-57 reduced all abrupt withdrawal signs (ie, platform jumping, paw flutters, head shakes, diarrhea, and total body weight loss), but did not elicit any cannabimimetic side effects. In addition, JZL184 or PF-3845 blocked naloxone-precipitated hypersecretion in morphine-dependent small intestinal tissue. Collectively, these results are the first to show that endocannabinoid catabolic enzyme inhibitors reduce abrupt withdrawal in morpine-dependent mice and are effective in a novel in vitro model of opioid withdrawal. More generally, these findings support the idea that joint MAGL and FAAH inhibition represents a promising approach for the treatment of opioid dependence.

Increases in αCaMKII phosphorylated on Thr286 in the nucleus accumbens shell but not the core during priming-induced reinstatement of morphine-seeking in rats.

Addiction is a pathological usurpation of the neural processes that normally serve reward-related learning and memory. Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) is an important molecule involved in the mechanisms of learning and memory, suggesting its roles in drug addiction. In this study, we detected the changes of CaMKII protein levels in the nucleus accumbens (NAc), a key nucleus involved in drug-reward, during the reinstatement of morphine-seeking behavior with animal model of morphine self-administration in rats. Moreover, considering that the NAc is also involved in the natural reward-related learning and memory, we detected the changes of CaMKII protein levels in the NAc during the reinstatement of natural reward-seeking with animal model of saccharin self-administration as a control. We found that the level of αCaMKII phosphorylated on Thr286 increased in the NAc shell subregion but not the NAc core during the reinstatement of morphine-seeking, compared with that after extinction. However, during the reinstatement of saccharin-seeking, the protein level of αCaMKII phosphorylated on Thr286 did not change in the NAc shell. Surprisingly, both αCaMKII phosphorylated on Thr286 and ßCaMKII phosphorylated on Thr287 decreased in the NAc core during the reinstatement of saccharin-seeking. These results suggest that increased phosphorylation of CaMKII (Thr286) in the NAc shell is involved in the relapse to opioids-seeking and the mechanisms underlying the reinstatement of morphine-seeking are different from those involved in the reinstatement of natural reward-seeking.

Mice deficient in endothelin-converting enzyme-2 exhibit abnormal responses to morphine and altered peptide levels in the spinal cord.

An increasing body of evidence suggests that endothelin-converting enzyme-2 (ECE-2) is a non-classical neuropeptide processing enzyme. Similar to other neuropeptide processing enzymes, ECE-2 exhibits restricted neuroendocrine distribution, intracellular localization, and an acidic pH optimum. However, unlike classical neuropeptide processing enzymes, ECE-2 exhibits a non-classical cleavage site preference for aliphatic and aromatic residues. We previously reported that ECE-2 cleaves a number of neuropeptides at non-classical sites in vitro; however its role in peptide processing in vivo is poorly understood. Given the recognized roles of neuropeptides in pain and opiate responses, we hypothesized that ECE-2 knockout (KO) mice might show altered pain and morphine responses compared with wild-type mice. We find that ECE-2 KO mice show decreased response to a single injection of morphine in hot-plate and tail-flick tests. ECE-2 KO mice also show more rapid development of tolerance with prolonged morphine treatment and fewer signs of naloxone-precipitated withdrawal. Peptidomic analyses revealed changes in the levels of a number of spinal cord peptides in ECE-2 KO as compared to wild-type mice. Taken together, our findings suggest a role for ECE-2 in the non-classical processing of spinal cord peptides and morphine responses; however, the precise mechanisms through which ECE-2 influences morphine tolerance and withdrawal remain unclear.

Protein kinase C phosphorylates the cAMP response element binding protein in the hypothalamic paraventricular nucleus during morphine withdrawal.

BACKGROUND AND PURPOSE: Exposure to drugs of abuse or stress results in adaptation in the brain involving changes in gene expression and transcription factors. Morphine withdrawal modulates gene expression through various second-messenger signal transduction systems. Here, we investigated changes in activation of the transcription factor, cAMP-response element binding protein (CREB), in the hypothalamic paraventricular nucleus (PVN) and the kinases that may mediate the morphine withdrawal-triggered activation of CREB and the response of the hypothalamic-pituitary-adrenocortical (HPA) axis after naloxone-induced morphine withdrawal. EXPERIMENTAL APPROACH: The effects of morphine dependence and withdrawal, phosphorylated CREB (pCREB), corticotrophin-releasing factor (CRF) expression in the PVN and HPA axis activity were measured using immunoblotting, immunohistochemistry and radioimmunoassay in controls and in morphine-dependent rats, withdrawn with naloxone and pretreated with vehicle, calphostin C, chelerythrine (inhibitors of protein kinase C (PKC) or SL-327 [inhibitor of extracellular signal regulated kinase (ERK) kinase]. In addition, changes in PKCα and PKCγ immunoreactivity were measured after 60 min of withdrawal. KEY RESULTS: In morphine-withdrawn rats, pCREB immunoreactivity was increased within CRF immunoreactive neurons in the PVN and plasma corticosterone levels were raised. SL-327, at doses that reduced the augmented pERK levels in the PVN, did not attenuate the rise in pCREB immunoreactivity or plasma corticosterone secretion. In contrast, PKC inhibition reduced the withdrawal-triggered rise in pCREB, pERK1/2 and corticosterone secretion. CONCLUSIONS AND IMPLICATIONS: PKC mediated, in part, both CREB activation and the HPA response to morphine withdrawal. The ERK kinase/ERK pathway might not be necessary for either activation of CREB or HPA axis hyperactivity.

[Comparative characteristics of glucose metabolism in the liver of rats under acute alcohol and morphine intoxication].

The comparative analysis effect of acute alcohol and morphine intoxications on rats on hepatic glycolysis and pentose phosphate pathway was done. The dose-dependent inhibitory effect of ethanol on activity of limiting enzymes of these metabolic ways, as well as anaerobic reorientation of glucose metabolism was recognised with the increase of the dose of the intake alcohol. Morfine (10 mg/kg) activated enymes of glycolysis and pentose phosphate pathway, but in contrast to ethanol it did not influence these parameters at the dose 20 or 40 mg/kg.

Ameliorating effect of histamine on impairment of cued fear extinction induced by morphine withdrawal in histidine decarboxylase gene knockout mice.

AIM: Histamine plays an important role in morphine addiction and memory-dependent behavior. However, little is known about the effect of histamine on the impairment of memory after morphine withdrawal. This study was designed to investigate the effect of histamine on memory impairment induced by morphine withdrawal in histidine decarboxylase knockout (HDC-KO) and wild-type (WT) mice. METHODS: WT and HDC-KO mice were given subcutaneous morphine or saline twice daily for 5 consecutive days. The mice received a cued or contextual fear conditioning session 7 days after the last injection. During subsequent days, mice received 4 cued or contextual extinction sessions (one session per day). Western blot was used to assess extracellular signal-regulated kinase (ERK) phosphorylation in the amygdala and hippocampus. RESULTS: Morphine withdrawal did not affect the acquisition of cued or contextual fear responses. It impaired cued but not contextual fear extinction. The acquisition of cued and contextual fear responses was accelerated in HDC-KO mice. Histamine deficiency aggravated the impairment of cued fear extinction induced by morphine withdrawal, whereas histamine (icv, 5 µg/mouse) reversed this effect. Morphine withdrawal decreased ERK phosphorylation in the amygdala after cued fear extinction, especially in HDC-KO mice. CONCLUSION: These results suggest that morphine withdrawal specifically impairs cued fear extinction and histamine ameliorates this impairment. Its action might be mediated by the modulation of ERK phosphorylation in the amygdala. Histamine should be explored for possible roles in the prevention or treatment of morphine abuse and relapse.

Valproate attenuates the development of morphine antinociceptive tolerance.

Morphine is a potent opioid analgesic. Repeated administration of morphine induces tolerance, thus reducing the effectiveness of analgesic treatment. Although some adjuvant analgesics can increase morphine analgesia, the precise molecular mechanism behind their effects remains unclear. Opioids bind to the mu opioid receptor (MOR). Morphine tolerance may be derived from alterations in the intracellular signal transduction after MOR activation. Chronic morphine treatment activates glycogen synthase kinase 3ß (GSK3ß), whose inhibition diminishes morphine tolerance. Valproate is widely prescribed as an anticonvulsant and a mood stabilizer for bipolar disorders because it increases the amount of γ-aminobutyric acid (GABA) in the central nervous system. Although the activation of GABAergic neurons may be responsible for the chief pharmacologic effect of valproate, recent studies have shown that valproate also suppresses GSK3ß activity. We examined the effect of valproate on the development of morphine antinociceptive tolerance in a mouse model of thermal injury. Mice were treated with morphine alone or with morphine and valproate twice daily for 5 days. The resulting antinociceptive effects were assessed using a hot plate test. While mice treated with morphine developed tolerance, co-administration of valproate attenuated the development of tolerance and impaired the activation of GSK3ß in mice brains. Valproate alone did not show analgesic effects; nevertheless, it functioned as an adjuvant analgesic to prevent the development of morphine tolerance. These results suggest that the modulation of GSK3ß activity by valproate may be useful and may play a role in the prevention of morphine tolerance.

The extinction of morphine-induced conditioned place preference by histone deacetylase inhibition.

Recent evidence suggests that epigenetic mechanisms have an important role in the development of addictive behavior. However, little is known about the role of epigenetic mechanisms in the extinction of morphine-induced behavioral changes. In this study, we will examine the effect of histone deacetylase (HDAC) inhibitors on extinction of morphine-induced conditioned place preference (CPP). To facilitate extinction, rats will be administered an HDAC inhibitor (HDACi) following nonreinforced exposure to the conditioned context. To measure persistence, rats were subject to a reinstatement test using 3 mg/kg dose of morphine. To exclude the effect of repeated NaBut injections themselves on morphine-CPP in the absence of extinction session, rats received injection of either NaBut or vehicle for 8 days. We found that HDAC inhibition during nonconfined extinction or confined extinction consolidation can facilitate extinction of morphine-induced CPP. We also showed that the extinction of drug seeking via HDAC inhibition modulates extinction learning such that reinstatement behavior is significantly attenuated. There is no effect of repeated NaBut injections themselves on morphine-CPP in the absence of extinction session. In conclusion, our results extend earlier reports on the ability of HDACi to modify the behavioral effects of drugs of abuse. Our increasing understanding of these epigenetic mechanisms will provide key answers to basic processes in drug addiction and hopefully provide insight into designing improved treatments for drug addiction.

[Anti-alcoholic and anti-narcotic action of Methylobacteriim extorquens UCM B-3368].

The paper deals with action efficiency of microbial biomass on characteristic indicators at alcohol and morphine organism intoxication. The investigated microbial biomass affects the regulatory biochemical and physiological systems in experimental animals, normalizes activity of alcohol dehydrogenase and aldehide dehydrogenase, as well as the content of dophamine, disturbed under the effect of alcohol and morphine. Thus, the organism intoxication decreases. Except for the specific action, the above microbial biomass can be a source of protein, aminoacids, vitamins, microelements. So, the microbial preparation, made on its basis, can be used for the treatment of alcohol and morphine dependence in a form of biologically active dope. Thus the microbial drug intended for treatment of alcohol and opium dependence has been developed. One of its action mechanisms is based on the microorganisms capacity to transform alcohols and aldehides, owing to availability of alcohol and aldehide dehydrogenase, other its action mechanisms are at the stage of investigation.

Delayed, context- and dopamine D1 receptor-dependent activation of ERK in morphine-sensitized mice.

Exposure to cues previously associated with intake of substances of abuse can promote drug related responses. In this study, we have examined the effect of exposure to a drug-associated context on the expression of morphine psychomotor sensitization. We show that sensitization is markedly increased in mice examined 4 weeks after the last morphine injection. In addition, this incubation period confers to the environment paired with morphine the ability to increase ERK phosphorylation in the shell (but not the core) of the nucleus accumbens. Using transgenic mice with enhanced green fluorescent protein (EGFP) expression under the control of the dopamine D1 receptor (D1R) (Drd1a-EGFP) or D2 receptor promoter (Drd2-EGFP) we show that context-dependent ERK phosphorylation is restricted to D1R-expressing medium spiny neurons. Furthermore, this effect depends on D1R activation. These data show that, following repeated morphine injections, a drug-free period induces context-dependent phosphorylation of ERK in a specific population of neurons within the nucleus accumbens shell. This activation is associated to enhanced psychomotor sensitization and may be implicated in context-elicited drug seeking induced by repeated exposure to drugs of abuse.

The PKs PKA and ERK 1/2 are involved in phosphorylation of TH at Serine 40 and 31 during morphine withdrawal in rat hearts.

BACKGROUND AND PURPOSE: 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. EXPERIMENTAL APPROACH: 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. KEY RESULTS: 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. CONCLUSIONS AND IMPLICATIONS: 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.

Pre-treatment with a PKC or PKA inhibitor prevents the development of morphine tolerance but not physical dependence in mice.

We previously demonstrated that intracerebroventricular (i.c.v.) administration of protein kinase C (PKC) or protein kinase A (PKA) inhibitors reversed morphine antinociceptive tolerance in 3-day morphine-pelleted mice. The present study aimed at evaluating whether pre-treating mice with a PKC or PKA inhibitor prior to pellet implantation would prevent the development of morphine tolerance and physical dependence. Antinociception was assessed using the warm-water tail immersion test and physical dependence was evaluated by quantifying/scoring naloxone-precipitated withdrawal signs. While drug-naïve mice pelleted with a 75 mg morphine pellet for 3 days developed a 5.8-fold tolerance to morphine antinociception, mice pre-treated i.c.v. with the PKC inhibitors bisindolylmaleimide I, Go-7874 or Go-6976, or with the myristoylated PKA inhibitor, PKI-(14-22)-amide failed to develop any tolerance to morphine antinociception. Experiments were also conducted to determine whether morphine-pelleted mice were physically dependent when pre-treated with PKC or PKA inhibitors. The same inhibitor doses that prevented morphine tolerance were evaluated in other mice injected s.c. with naloxone and tested for precipitated withdrawal. The pre-treatment with PKC or PKA inhibitors failed to attenuate or block the signs of morphine withdrawal including jumping, wet-dog shakes, rearing, forepaw tremor, increased locomotion, grooming, diarrhea, tachypnea and ptosis. These data suggest that elevations in the activity of PKC and PKA in the brain are critical to the development of morphine tolerance. However, it appears that tolerance can be dissociated from physical dependence, indicating a role for PKC and PKA to affect antinociception but not those signs mediated through the complex physiological processes of withdrawal.

Implication of endogenous beta-endorphin in the inhibition of the morphine-induced rewarding effect by the direct activation of spinal protein kinase C in mice.

It has often been proposed that opioid addiction does not arise as a consequence of opioid treatment for pain. Recently, we demonstrated that activated protein kinase C (PKC) in the spinal cord associated with chronic pain-like hyperalgesia suppressed the morphine-induced rewarding effect in mice. In the present study, we investigated whether a gene deletion for an endogenous mu-opioid peptide beta-endorphin could affect pain-like behavior and the suppression of the morphine-induced rewarding effect by the direct activation of PKC in the spinal cord. We found that activation of spinal PKC by intrathecal (i.t.) treatment with phorbol 12,13-dibutyrate (PDBu), a specific PKC activator, caused thermal hyperalgesia, pain-like behaviors and suppression of the morphine-induced rewarding effect. This suppression of morphine reward was eliminated in mice that lacked beta-endorphin. In contrast, thermal hyperalgesia and pain-like behaviors were not affected in beta-endorphin knockout mice. These results suggest that the activation of PKC in the spinal cord may play an essential role in the suppression of the morphine-induced rewarding effect in mice with neuropathic pain through the constant release of beta-endorphin.

[Changes of adenylate cyclase on cerebral regions related to mophine dependence in rats].

OBJECTIVE: To observe the changes of adenylate cyclase(AC) on cerebral regions related to morphine dependence in rats and investigate the relationship between the enzymological changes and the mechanism of morphine dependence. METHODS: The technique of enzyme-histochemistry was used to detect the variations of AC of special seven cerebral regions including frontalis cortex, lenticula, corpus amygdaloideun, substantia nigra, hippocampus, periaqueductal gray and locus coerleus in morphine dependent rats. The enzymological changes were observed by optical microscope. Changes of gray degree of these cerebral regions were also observed by using the image analysis system. RESULTS: Compared with those in control group, the contents of AC in morphine dependent groups were increased. CONCLUSION: The contents of AC are increase in those regions. The mechanism of morphine dependence close related to the increasing of AC. The correlation of the mechanism of morphine dependence and up-regulation of AC/cAMP-PKA system is discussed.

[Activation of the spinal extracellular signal-regulated kinase is involved in morphine dependence and naloxone-precipitated withdrawal response].

Extracellular signal-regulated kinase (ERK), a mitogen-activated protein kinase (MAPK), transduces a broad range of extracellular stimuli into diverse intracellular responses. It has been reported that ERK is involved in the modulation of nociceptive information and central sensitization produced by intense noxious stimuli or peripheral tissue inflammation. Our previous studies showed that the spinal neurons sensitization was involved in morphine withdrawal response. This study was to investigate the role of the spinal ERK in morphine dependence and naloxone-precipitated withdrawal response. To set up morphine-dependent model, rats were subcutaneously injected with morphine (twice a day, for 5 d). The dose of morphine was 10 mg/kg on the first day and was increased by 10 mg/kg each day. On day 6, 4 h after the injection of morphine (50 mg/kg), morphine withdrawal syndrome was precipitated by an injection of naloxone (4 mg/kg, i.p.). Using anti-phospho-ERK (pERK) antibody, the time course of pERK expression was detected by Western blot. U0126, a mitogen-activated protein kinase kinase (MEK) inhibitor, or phosphorothioate-modified antisense oligonucleotides (ODN) was intrathecally injected 30 min or 36, 24 and 12 h before naloxone-precipitated withdrawal. The scores of morphine withdrawal symptom and morphine withdrawal-induced allodynia were observed. One hour after naloxone-precipitated withdrawal, pERK expression in the spinal dorsal horn was assessed by immunohistochemical analysis and Western blot was used to detect the expression of cytosolic and nuclear fraction of pERK in the rat spinal cord. The results showed that the expression of cytosolic and nuclear fraction of pERK, not non-phospho-ERK, in the spinal cord was gradually increased following the injection of morphine. When morphine withdrawal was precipitated with naloxone, the expression of the spinal pERK further increased. Intrathecal administration of U0126 or antisense ODN against ERK decreased the scores of morphine withdrawal, attenuated morphine withdrawal-induced allodynia and also inhibited the increase of pERK expression in the spinal cord of morphine withdrawal rats. These results suggest that activation of the spinal ERK is involved in morphine-dependent and naloxone-precipitated withdrawal response.

Implication of cyclin-dependent kinase 5 in the development of psychological dependence on and behavioral sensitization to morphine.

In the present study, we investigated the role of cyclin-dependent kinase 5 (cdk5) in the brain dynamics changed by repeated in vivo treatment with morphine. The level of phosphorylated-cdk5 was significantly increased in the cingulate cortex of mice showing the morphine-induced rewarding effect. Under these conditions, roscovitine, a cdk5 inhibitor, given intracerebroventricularly (i.c.v.) caused a dose-dependent and significant inhibition of the morphine-induced rewarding effect. In addition, the dose-response effect of the morphine-induced rewarding effect was dramatically attenuated in cdk5 heterozygous (+/-) knockout mice. Furthermore, the development of behavioral sensitization by intermittent administration of morphine was virtually abolished in cdk5 (+/-) mice. These findings suggest that the induction and/or activation of cdk5 are implicated in the development of psychological dependence on morphine.

[The development of research on enzymes related to morphine-dependent].

The mechanism of morphine dependent is a complex Procedure. It involves in many complex mechanisms such as the ultra-structure of synapse of special brain areas, neurotransmitter, enzymology, and so on. These mechanisms have closely correlation. In this paper we reveiwed the development in enzymological mechanism of morphine dependent enzymes including protein kinase (PK), nitric oxide synthase (NOS), superoxide dismutase (SOD), adenylate cyclase (AC), Succinate dehydrogenase (SDH)and 3beta-Hydroxy steroid dehydrogenase (3beta-HSD).