Glutamatergic neurons in ventral pallidum modulate heroin addiction via epithalamic innervation in rats.

Glutamatergic neurons in ventral pallidum (VPGlu) were recently reported to mediate motivational and emotional behavior, but its role in opioid addiction still remains to be elucidated. In this study we investigated the function of VPGlu in the context-dependent heroin taking and seeking behavior in male rats under the ABA renewal paradigm. By use of cell-type-specific fiber photometry, we showed that the calcium activity of VPGlu were inhibited during heroin self-administration and context-induced relapse, but activated after extinction in a new context. The drug seeking behavior was accompanied by the decreased calcium signal of VPGlu. Chemogenetic manipulation of VPGlu bidirectionally regulated heroin taking and seeking behavior. Anterograde tracing showed that the lateral habenula, one of the epithalamic structures, was the major output region of VPGlu, and its neuronal activity was consistent with VPGlu in different phases of heroin addiction and contributed to the motivation for heroin. VPGlu axon terminals in LHb exhibited dynamic activity in different phases of heroin addiction. Activation of VPGlu-LHb circuit reduced heroin seeking behavior during context-induced relapse. Furthermore, the balance of excitation/inhibition from VP to LHb was shifted to enhanced glutamate transmission after extinction of heroin seeking motivation. Overall, the present study demonstrated that the activity of VPGlu was involved in the regulation of heroin addiction and identified the VPGlu-LHb pathway as a potential intervention to reduce heroin seeking motivation.

Electroacupuncture Ameliorates Depression-Like Behaviors Comorbid to Chronic Neuropathic Pain via Tet1-Mediated Restoration of Adult Neurogenesis.

Although electroacupuncture (EA) stimulation is a widely used therapy for chronic pain and comorbid psychiatric disorders, its long-term effects on chronic neuropathic pain-induced depression and the underlying mechanisms remain elusive. In the present study, we found that EA stimulation was able to restore adult neurogenesis in the ventral dentate gyrus (DG), by both increasing neuronal differentiation and restoring the normal morphology of newborn dendrites, in mice with spared nerve injury surgery. By ablating the Nestin+ neural stem cells (NSCs) via diphtheria toxin fragment A expression, we further proved that neurogenesis in the ventral DG was crucial to the long-term, but not the immediate antidepressant effect of EA, nor was it associated with nociception. Furthermore, we found that the restoration of neurogenesis was dependent on Tet1-mediated epigenetic modification upon EA treatment. Tet1 could bind to the promoter of the Prox1 gene, thus catalyzing its demethylation and facilitating its expression, which finally contributed to the restoration of neurogenesis and amelioration of depression-like behaviors induced by chronic neuropathic pain. Thus, we conclude that EA stimulation restores inhibited Tet1 expression in hippocampal NSCs of mice with chronic neuropathic pain, and increased Tet1 expression ameliorates hypermethylation of Prox1 and restores normal adult neurogenesis in the ventral DG, which contributes to the long-term antidepressant effect of EA.

The Agonist of Adenosine A1 Receptor Induced Desensitization of delta Opioid receptor-mediated Raf-1/MEK/ERK Signaling by Feedback Phosphorylation of Raf-1-Ser289/296/301.

Our previous study found that activation of adenosine A1 receptor (A1R) induced phosphorylation of delta opioid receptor (DOR) and desensitization of its downstream signaling molecules, cAMP and Akt. To further investigate the effect of A1R agonist on DOR signaling and the underlying mechanism, we examined the effect of A1R activation upon binding of its agonist N6-cyclohexyl-adenosine (CHA) on DOR-mediated Raf-1/MEK/ERK activation, and found that prolonged CHA exposure resulted in downregulation of DOR-mediated Raf-1/MEK/ERK signaling pathway. CHA-treatment time dependently attenuated Raf-1-Ser338 phosphorylation induced by [D-Pen2,5] enkephalin (DPDPE), a specific agonist of DOR, and further caused downregulation of the Raf-1/MEK/ERK signaling pathway activated by DOR agonist. Moreover, CHA exposure time-dependently induced the phosphorylation of Raf-1-Ser289/296/301, the inhibitory phosphorylation sites that were regulated by negative feedback, thereby inhibiting activation of the MEK/ERK pathway, and this effect could be blocked by MEK inhibitor U0126. Finally, we proved that the heterologous desensitization of the Raf-1/MEK/ERK cascade was essential in the regulation of anti-nociceptive effect of DOR agonists by confirming that such effect was inhibited by pretreatment of CHA. Therefore, we conclude that the activation of A1R inhibits DOR-mediated MAPK signaling pathway via heterologous desensitization of the Raf-1/MEK/ERK cascade, which is a result of ERK-mediated Raf-1-Ser289/296/301 phosphorylation mediated by activation of A1R.

Amygdala dynorphin/κ opioid receptor system modulates depressive-like behavior in mice following chronic social defeat stress.

Major depression disorder is a severe and recurrent neuropsychological disorder characterized by lowered mood and social activity and cognitive impairment. Owing to unclear molecular mechanisms of depression, limited interventions are available in clinic. In this study we investigated the role of dynorphin/κ opioid receptor system in the development of depression. Mice were subjected to chronic social defeat stress for 14 days. Chronic social defeat stress induced significant social avoidance in mice characterized by decreased time duration in the interaction zone and increased time duration in the corner zone. Pre-administration of a κ opioid receptor antagonist norBNI (10 mg/kg, i.p.) could prevent the development of social avoidance induced by chronic social defeat stress. Social avoidance was not observed in κ opioid receptor knockout mice subjected to chronic social defeat stress. We further revealed that social defeat stress activated c-fos and ERK signaling in the amygdala without affecting the NAc, hippocampus and hypothalamus, and ERK activation was blocked by systemic injection of norBNI. Finally, the expression of dynorphin A, the endogenous ligand of κ opioid receptor, was significantly increased in the amygdala following social defeat stress; microinjection of norBNI into the amygdala prevented the development of depressive-like behaviors caused by social defeat stress. The present study demonstrates that upregulated dynorphin/κ opioid receptor system in the amygdala leads to the emergence of depression following chronic social defeat stress, and sheds light on κ opioid receptor antagonists as potential therapeutic agents for the prevention and treatment of depression following chronic stress.

Dezocine as a potent analgesic: overview of its pharmacological characterization.

Dezocine, a synthetic opioid, introduced in 1970s as an analgesic, was redeveloped for relieving moderate to severe pain by Yangtze River Pharmaceutical Group in China in 2009. To date, dezocine occupies 45% of China's opioid analgesic market. Along with dezocine being a dominated painkiller, a certain amount of research was conducted to elucidate dezocine's action. In this review we summarize the current knowledge on the receptor, preclinical and clinical pharmacology of dezocine. Briefly, preclinical data show that dezocine is effective under varying pain conditions, particularly chronic neuropathic pain and cancer pain, through activation of opioid receptors, and inhibition of norepinephrine reuptake. Clinical data establish the effectiveness of dezocine either as a primary analgesic for postoperative pain management or a supplement for balanced analgesia. The receptor profile of dezocine is different from known pure µ agonists, and allows it to be used in combination with other opioids for additivity in efficacy or lower incidence of adverse effects.

Discovery of 5-Benzylidene-2-phenyl-1,3-dioxane-4,6-diones as Highly Potent and Selective SIRT1 Inhibitors.

SIRT1, a member of the sirtuin family, catalyzes the deacetylation of proteins with the transformation of NAD+ into nicotinamide and 2'-O-acetyl-ADP-ribose. Selective SIRT1/2 inhibitors have potential application in the chemotherapy of colorectal carcinoma, prostate cancer, and myelogenous leukemia. Here we identified novel SIRT1 inhibitors with the scaffold of 5-benzylidene-2-phenyl-1,3-dioxane-4,6-dione. The most potent inhibitor 12n displayed an IC50 of 460 nM and a selectivity for SIRT1 over SIRT2, SIRT3, and SIRT5 of 113.5-, 254.3-, and 10.83-fold, respectively. It did not affect the activity of SIRT6. To elucidate the inhibitory mechanism, we determined the inhibition type of the inhibitor by enzyme kinetic analysis, showing that the inhibitor was competitive to the acetyl peptide and noncompetitive to NAD+. Further, the interaction of the inhibitor in SIRT1 was studied by using molecular docking, which was validated by the structure-activity relationship analysis of the inhibitors and the site-directed mutagenesis of SIRT1. Consistent with the in vitro assays, the inhibitors increased the acetylation level of p53 in a concentration-dependent manner in cells.

Src-dependent phosphorylation of µ-opioid receptor at Tyr336 modulates opiate withdrawal.

Opiate withdrawal/negative reinforcement has been implicated as one of the mechanisms for the progression from impulsive to compulsive drug use. Increase in the intracellular cAMP level and protein kinase A (PKA) activities within the neurocircuitry of addiction has been a leading hypothesis for opiate addiction. This increase requires the phosphorylation of µ-opioid receptor (MOR) at Tyr336 by Src after prolonged opiate treatment in vitro Here, we report that the Src-mediated MOR phosphorylation at Tyr336 is a prerequisite for opiate withdrawal in mice. We observed the recruitment of Src in the vicinity of MOR and an increase in phosphorylated Tyr336 (pY336) levels during naloxone-precipitated withdrawal. The intracerebroventricular or stereotaxic injection of a Src inhibitor (AZD0530), or Src shRNA viruses attenuated pY336 levels, and several somatic withdrawal signs. This was also observed in Fyn-/- mice. The stereotaxic injection of wild-type MOR, but not mutant (Y336F) MOR, lentiviruses into the locus coeruleus of MOR-/- mice restored somatic withdrawal jumping. Regulating pY336 levels during withdrawal might be a future target for drug development to prevent opiate addictive behaviors.

Heteromers of µ opioid and dopamine D1 receptors modulate opioid-induced locomotor sensitization in a dopamine-independent manner.

BACKGROUND AND PURPOSE: Exposure to opiates induces locomotor sensitization in rodents, which has been proposed to correspond to the compulsive drug-seeking behaviour. Numerous studies have demonstrated that locomotor sensitization can occur in a dopamine transmission-independent manner; however, the underlying mechanisms are unclear. EXPERIMENTAL APPROACH: Co-immunoprecipitation, BRET and cross-antagonism assays were used to demonstrate the existence of receptor heterodimers. Function of heterodimers was evaluated by behavioural studies of locomotor sensitization. KEY RESULTS: The dopamine D1 receptor antagonist SCH23390 antagonized the signalling initiated by stimulation of µ opioid receptors with agonists in transfected cells expressing two receptors and in striatal tissues from wild-type but not D1 receptor knockout (KO) mice, suggesting that SCH23390 modified µ receptor function via receptor heteromers, as the ability of an antagonist of one of the receptors to inhibit signals originated by stimulation of the partner receptor was a characteristic of receptor heteromers. The existence of µ receptor-D1 receptor heterodimers was further supported by biochemical and biophysical assays. In vivo, when dopamine release was absent (by destruction of the dopaminergic projection from the ventral tegmental area to the striatum), SCH23390 still significantly inhibited µ receptor agonist-induced behavioural responses in rats. Additionally, we demonstrated that D1 or µ receptor KO mice and thus unable to form µ receptor-D1 receptor heterodimers, failed to show locomotor sensitization to morphine. CONCLUSION AND IMPLICATIONS: Our results suggest that µ receptor-D1 receptor heterodimers may be involved in the dopamine-independent expression of locomotor sensitization to opiates.

The neuroprotective effect of memantine on methamphetamine-induced cognitive deficits.

Repeated exposure to methamphetamine (METH) can cause severe neurotoxicity to the cortical neurons. In the present study, we investigated the effect of METH on cognitive function deficits, and determined the neuroprotective effects of memantine (MEM) on memory impairment induced by METH. The protein levels of Bcl-2 and cleaved caspase-3 in prefrontal cortex (PFC) were further examined to exploring the underlying mechanism. We found that repeated METH administration impaired long term (24h) memory retention without affecting short term (5min) memory retention. Co-administration of MEM with METH before training session significantly improved METH-induced cognitive function. METH significantly decreased expression level of Bcl-2 and increased expression level of cleaved caspase-3 in the PFC. The changes can be prevented by MEM pretreatment. Thus, these results demonstrated that MEM pretreatment reversed METH-induced changes of protein levels of apoptotic-related gene, and produced protective effects against METH-induced cognitive deficits, suggesting the effectiveness of MEM may be due to its anti-apoptotic activity.

Dissociative role for dorsal hippocampus in mediating heroin self-administration and relapse through CDK5 and RhoB signaling revealed by proteomic analysis.

Addiction is characterized by drug craving, compulsive drug taking and relapse, which is attributed to aberrant neuroadaptation in brain regions implicated in drug addiction, induced by changes in gene and protein expression in these regions after chronic drug exposure. Accumulating evidence suggests that the dorsal hippocampus (DH) plays an important role in mediating drug-seeking and drug-taking behavior and relapse. However, the molecular mechanisms underlying these effects of the DH are unclear. In the present study, we employed a label-free quantitative proteomic approach to analyze the proteins altered in the DH of heroin self-administering rats. A total of 4015 proteins were quantified with high confidence, and 361 proteins showed significant differences compared with the saline control group. Among them, cyclin-dependent kinase 5 (CDK5) and ras homolog family member B (RhoB) were up-regulated in rats with a history of extended access to heroin. Functionally, inhibition of CDK5 in the DH enhanced heroin self-administration, indicating that CDK5 signaling in the DH acts as a homeostatic compensatory mechanism to limit heroin-taking behavior, whereas blockade of the Rho-Rho kinase (ROCK) pathway attenuated context-induced heroin relapse, indicating that RhoB signaling in the DH is required for the retrieval (recall) of addiction memory. Our findings suggest that manipulation of CDK5 signaling in the DH may be essential in determining vulnerability to opiate taking, whereas manipulation of RhoB signaling in the DH may be essential in determining vulnerability to relapse. Overall, the present study suggests that the DH can exert dissociative effects on heroin addiction through CDK5 and RhoB signaling.

Role for engagement of ß-arrestin2 by the transactivated EGFR in agonist-specific regulation of δ receptor activation of ERK1/2.

BACKGROUND AND PURPOSE: ß-Arrestins function as signal transducers linking GPCRs to ERK1/2 signalling either by scaffolding members of ERK1/2s cascades or by transactivating receptor tyrosine kinases through Src-mediated release of transactivating factor. Recruitment of ß-arrestins to the activated GPCRs is required for ERK1/2 activation. Our previous studies showed that δ receptors activate ERK1/2 through a ß-arrestin-dependent mechanism without inducing ß-arrestin binding to the δ receptors. However, the precise mechanisms involved remain to be established. EXPERIMENTAL APPROACH: ERK1/2 activation by δ receptor ligands was assessed using HEK293 cells in vitro and male Sprague Dawley rats in vivo. Immunoprecipitation, immunoblotting, siRNA transfection, intracerebroventricular injection and immunohistochemistry were used to elucidate the underlying mechanism. KEY RESULTS: We identified a new signalling pathway in which recruitment of ß-arrestin2 to the EGFR rather than δ receptor was required for its role in δ receptor-mediated ERK1/2 activation in response to H-Tyr-Tic-Phe-Phe-OH (TIPP) or morphine stimulation. Stimulation of the δ receptor with ligands leads to the phosphorylation of PKCδ, which acts upstream of EGFR transactivation and is needed for the release of the EGFR-activating factor, whereas ß-arrestin2 was found to act downstream of the EGFR transactivation. Moreover, we demonstrated that coupling of the PKCδ/EGFR/ß-arrestin2 transactivation pathway to δ receptor-mediated ERK1/2 activation was ligand-specific and the Ser(363) of δ receptors was crucial for ligand-specific implementation of this ERK1/2 activation pathway. CONCLUSIONS AND IMPLICATIONS: The δ receptor-mediated activation of ERK1/2 is via ligand-specific transactivation of EGFR. This study adds new insights into the mechanism by which δ receptors activate ERK1/2.

Pharmacological characterization and therapeutic potential for the treatment of opioid abuse with ATPM-ET, an N-ethyl substituted aminothiazolomorphinan with κ agonist and µ agonist/antagonist activity.

We previously reported that the κ agonists with mixed µ activity could attenuate heroin self-administration with less potential to develop tolerance. The present study further investigated the effects of (-)-3-N-Ethylamino-thiazolo[5,4-b]-N-cyclopropylmethylmorphinan hydrochloride (ATPM-ET), a κ agonist and µ agonist/antagonist, on the acquisition and reinstatement of morphine-induced conditioned place preference (CPP), heroin self-administration and heroin-primed reinstatement of drug-seeking behavior. We found that ATPM-ET produced a longer duration of potent antinociceptive effects with less side effect of sedation. More importantly, ATPM-ET attenuated the acquisition of morphine-induced CPP, without affecting the reinstatement of morphine CPP. Furthermore, ATPM-ET significantly inhibited heroin self-administration and the reinstatement of heroin primed drug-seeking behavior. Taken together, ATPM-ET, a novel κ agonist and µ agonist/antagonist may have utility for the treatment of drug dependence.

Serine 363 of the {delta}-opioid receptor is crucial for adopting distinct pathways to activate ERK1/2 in response to stimulation with different ligands.

Distinct opioid receptor agonists have been proved to induce differential patterns of ERK activation, but the underlying mechanisms remain unclear. Here, we report that Ser363 in the δ-opioid receptor (δOR) determines the different abilities of the δOR agonists DPDPE and TIPP to activate ERK by G-protein- or ß-arrestin-dependent pathways. Although both DPDPE and TIPP activated ERK1/2, they showed different temporal, spatial and desensitization patterns of ERK activation. We show that that DPDPE employed G protein as the primary mediator to activate the ERK cascade in an Src-dependent manner, whereas TIPP mainly adopted a ß-arrestin1/2-mediated pathway. Moreover, we found that DPDPE gained the capacity to adopt the ß-arrestin1/2-mediated pathway upon Ser363 mutation, accompanied by the same pattern of ERK activation as that induced by TIPP. Additionally, we found that TIPP- but not DPDPE-activated ERK could phosphorylate G-protein-coupled receptor kinase-2 and ß-arrestin1. However, such functional differences of ERK disappeared with the mutation of Ser363. Therefore, the present study reveals a crucial role for Ser363 in agonist-specific regulation of ERK activation patterns and functions.

Adenosine A(1) receptor agonist N(6)-cyclohexyl-adenosine induced phosphorylation of delta opioid receptor and desensitization of its signaling.

AIM: To define the effect of adenosine A(1) receptor (A(1)R) on delta opioid receptor (DOR)-mediated signal transduction. METHODS: CHO cells stably expressing HA-tagged A(1)R and DOR-CFP fusion protein were used. The localization of receptors was observed using confocal microscope. DOR-mediated inhibition of adenylyl cyclase was measured using cyclic AMP assay. Western blots were employed to detect the phosphorylation of Akt and the DOR. The effect of A(1)R agonist N(6)-cyclohexyladenosine (CHA) on DOR down-regulation was assessed using radioligand binding assay. RESULTS: CHA 1 micromol/L time-dependently attenuated DOR agonist [D-Pen(2,5)]enkephalin (DPDPE)-induced inhibition of intracellular cAMP accumulation with a t(1/2)=2.56 (2.09-3.31) h. Pretreatment with 1 micromol/L CHA for 24 h caused a right shift of the dose-response curve of DPDPE-mediated inhibition of cAMP accumulation, with a significant increase in EC(50) but no change in E(max). Pretreatment with 1 micromol/L CHA for 1 h also induced a significant attenuation of DPDPE-stimulated phosphorylation of Akt. Moreover, CHA time-dependently phosphorylated DOR (Ser363), and this effect was inhibited by A(1)R antagonist 1,3-Dipropyl-8-cyclopentylxanthine (DPCPX) but not by DOR antagonist naloxone. However, CHA failed to produce the down-regulation of DOR, as neither receptor affinity (K(d)) nor receptor density (B(max)) of DOR showed significant change after chronic CHA exposure. CONCLUSION: Activation of A(1)R by its agonist caused heterologous desensitization of DOR-mediated inhibition of intracellular cAMP accumulation and phosphorylation of Akt. Activation of A(1)R by its agonist also induced heterologous phosphorylation but not down-regulation of DOR.

Chronic morphine treatment impaired hippocampal long-term potentiation and spatial memory via accumulation of extracellular adenosine acting on adenosine A1 receptors.

Chronic exposure to opiates impairs hippocampal long-term potentiation (LTP) and spatial memory, but the underlying mechanisms remain to be elucidated. Given the well known effects of adenosine, an important neuromodulator, on hippocampal neuronal excitability and synaptic plasticity, we investigated the potential effect of changes in adenosine concentrations on chronic morphine treatment-induced impairment of hippocampal CA1 LTP and spatial memory. We found that chronic treatment in mice with either increasing doses (20-100 mg/kg) of morphine for 7 d or equal daily dose (20 mg/kg) of morphine for 12 d led to a significant increase of hippocampal extracellular adenosine concentrations. Importantly, we found that accumulated adenosine contributed to the inhibition of the hippocampal CA1 LTP and impairment of spatial memory retrieval measured in the Morris water maze. Adenosine A(1) receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine significantly reversed chronic morphine-induced impairment of hippocampal CA1 LTP and spatial memory. Likewise, adenosine deaminase, which converts adenosine into the inactive metabolite inosine, restored impaired hippocampal CA1 LTP. We further found that adenosine accumulation was attributable to the alteration of adenosine uptake but not adenosine metabolisms. Bidirectional nucleoside transporters (ENT2) appeared to play a key role in the reduction of adenosine uptake. Changes in PKC-alpha/beta activity were correlated with the attenuation of the ENT2 function in the short-term (2 h) but not in the long-term (7 d) period after the termination of morphine treatment. This study reveals a potential mechanism by which chronic exposure to morphine leads to impairment of both hippocampal LTP and spatial memory.

Paradoxical relationship between RAVE (relative activity versus endocytosis) values of several opioid receptor agonists and their liability to cause dependence.

AIM: To examine the relationship between the RAVE (relative activity versus endocytosis) values of opiate agonists and their dependence liability by studying several potent analgesics with special profiles in the development of physical and psychological dependence. METHODS: The effects of (-)-cis-(3R,4S,2'R) ohmefentanyl (F9202), (+)-cis-(3R,4S,2'S) ohmefentanyl (F9204), dihydroetorphine (DHE) and morphine on [(35)S]GTP gamma S binding, forskolin-stimulated cAMP accumulation, and receptor internalization were studied in CHO cells stably expressing HA-tagged mu-opioid receptors (CHO-HA-MOR). cAMP overshoot in response to the withdrawal of these compound treatments was also tested. RESULTS: All four agonists exhibited the same rank order of activity in stimulation of [(35)S]GTP gamma S binding, inhibition of adenylyl cyclase (AC) and induction of receptor internalization: DHE>F9204>F9202>morphine. Based on these findings and the previous in vivo analgesic data obtained from our and other laboratories, the RAVE values of the four agonists were calculated. The rank order of RAVE values was morphine>F9202>F9204>DHE. For the induction of cAMP overshoot, the rank order was F9202>or=morphine>F9204>or=DHE. CONCLUSION: Taken in combination with previous findings of these compounds' liability to develop dependence, the present study suggests that the agonist with the highest RAVE value seems to have a relatively greater liability to develop psychological dependence relative to the agonist with the lowest RAVE value. However, the RAVE values of these agonists are not correlated with their probability of developing physical dependence or inducing cAMP overshoot, a cellular hallmark of dependence.

Initial experience of heroin use under a two-chained operant schedule influences drug-seeking behavior after one month of abstinence.

AIM: To evaluate the influence of an initial heroin experience under a modified two-chained training schedule on drug-seeking behavior after a long abstinence period. METHODS: Rats were trained to respond for intravenous heroin (120 microg/kg) under a heterogeneous chained schedule of reinforcement using different responses in the first and second links of the chain. Animals received low-frequency drug administration training for four days and were then subjected to one month of abstinence in their home cages. Heroin-seeking behavior induced by re-exposure to the first chain associated context or discriminative stimuli was assessed after abstinence. RESULTS: Almost all animals could acquire operant skills quickly under the two-chained schedule training for four days, as measured in first active response latency, travel speed and goal-box enter latency. Both first chain associated context and discriminative stimulus could reinstate heroin-seeking behavior after one month abstinence. CONCLUSION: These observations suggest that an early experience of drug use is sufficient to maintain heroin-seeking behavior even after a one month abstinence.

Chronic high-dose morphine treatment promotes SH-SY5Y cell apoptosis via c-Jun N-terminal kinase-mediated activation of mitochondria-dependent pathway.

Chronic high doses of morphine inhibit the growth of various human cancer cell lines. However, the mechanisms by which such high-dose morphine inhibits cell proliferation and induces cell death are not fully understood. Here we show that c-Jun N-terminal kinase (JNK) plays a pivotal role in high-dose morphine-induced apoptosis of SH-SY5Y cells in a mitochondria-dependent manner. Activation of JNK by morphine led to reactive oxygen species (ROS) generation via the mitochondrial permeability transition pore, because the mPTP inhibitor cyclosporin A significantly inhibited ROS generation. ROS in turn exerted feedback regulation on JNK activation, as shown by the observations that cyclosporin A and the antioxidant N-acetylcysteine significantly inhibited the phosphorylation of JNK induced by morphine. ROS-amplified JNK induced cytochrome c release and caspase-9/3 activation through enhancement of expression of the proapoptotic protein Bim and reduction of expression of the antiapoptotic protein Bcl-2. All of these effects of morphine could be suppressed by the JNK inhibitor SP600125 and N-acetylcysteine. The key role of the JNK pathway in morphine-induced apoptosis was further confirmed by the observation that decreased levels of JNK in cells transfected with specific small interfering RNA resulted in resistance to the proapoptotic effect of morphine. Thus, the present study clearly shows that morphine-induced apoptosis in SH-SY5Y cells involves JNK-dependent activation of the mitochondrial death pathway, and that ROS signaling exerts positive feedback regulation of JNK activity.

Pharmacological characterization of ATPM [(-)-3-aminothiazolo[5,4-b]-N-cyclopropylmethylmorphinan hydrochloride], a novel mixed kappa-agonist and mu-agonist/-antagonist that attenuates morphine antinociceptive tolerance and heroin self-administration behavior.

ATPM [(-)-3-amino-thiazolo[5,4-b]-N-cyclopropylmethylmorphinan hydrochloride] was found to have mixed kappa- and mu-opioid activity and identified to act as a full kappa-agonist and a partial mu-agonist by in vitro binding assays. The present study was undertaken to characterize its in vivo effects on morphine antinociceptive tolerance in mice and heroin self-administration in rats. ATPM was demonstrated to yield more potent antinociceptive effects than (-)U50,488H (trans-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl]benzeneacetamide). It was further found that the antinociceptive effects of ATPM were mediated by kappa- and mu-, but not delta-opioid, receptors. In addition to its agonist profile on the mu-receptor, ATPM also acted as a mu-antagonist, as measured by its inhibition of morphine-induced antinociception. It is more important that ATPM had a greater ratio of the ED(50) value of sedation to that of antinociception than (-)U50,488 (11.8 versus 3.7), indicative of a less sedative effect than (-)U50,488H. In addition, ATPM showed less potential to develop antinociceptive tolerance relative to (-)U50,488H and morphine. Moreover, it dose-dependently inhibited morphine-induced antinociceptive tolerance. Furthermore, it was found that chronic treatment of rats for 8 consecutive days with ATPM (0.5 mg/kg s.c.) produced sustained decreases in heroin self-administration. (-)U50,488H (2 mg/kg s.c.) also produced similar inhibitory effect. Taken together, our findings demonstrated that ATPM, a novel mixed kappa-agonist and mu-agonist/-antagonist, could inhibit morphine-induced antinociceptive tolerance, with less potential to develop tolerance and reduce heroin self-administration with less sedative effect. kappa-Agonists with some mu-activity appear to offer some advantages over selective kappa-agonists for the treatment of heroin abuse.

Morphine inhibits doxorubicin-induced reactive oxygen species generation and nuclear factor kappaB transcriptional activation in neuroblastoma SH-SY5Y cells.

Morphine is recommended as a first-line opioid analgesic in the pain management of cancer patients. Accumulating evidence shows that morphine has anti-apoptotic activity, but its impact on the therapeutic applications of antineoplastic drugs is not well known. The present study was undertaken to test the hypothesis that morphine might antagonize the pro-apoptotic activity of DOX (doxorubicin), a commonly used antitumour drug for the treatment of neuroblastoma, in cultured SH-SY5Y cells. In the present study we demonstrated that morphine suppressed DOX-induced inhibition of cell proliferation and programmed cell death in a concentration-dependent, and naloxone as well as pertussis toxin-irreversible, manner. Further studies showed that morphine inhibited ROS (reactive oxygen species) generation, and prevented DOX-mediated caspase-3 activation, cytochrome c release and changes of Bax and Bcl-2 protein expression. The antioxidant NAC (N-acetylcysteine) also showed the same effects as morphine on DOX-induced ROS generation, caspase-3 activation and cytochrome c release and changes in Bax (Bcl-2-associated X protein) and Bcl-2 protein expression. Additionally, morphine was found to suppress DOX-induced NF-kappaB (nuclear factor kappaB) transcriptional activation via a reduction of IkappaBalpha (inhibitor of nuclear factor kappaB) degradation. These present findings support the hypothesis that morphine can inhibit DOX-induced neuroblastoma cell apoptosis by the inhibition of ROS generation and mitochondrial cytochrome c release, as well as by blockade of NF-kappaB transcriptional activation, and suggests that morphine might have an impact on the antitumour efficiency of DOX.