Publications by authors named "Zhi-Qiang Chi"

34 Publications

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

Br J Pharmacol 2017 Sep 18;174(17):2842-2861. Epub 2017 Jul 18.

Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy of Science, Collaborative Innovation Center for Brain Science, Shanghai, China.

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 D 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 D 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-D 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 D or μ receptor KO mice and thus unable to form μ receptor-D receptor heterodimers, failed to show locomotor sensitization to morphine.

Conclusion And Implications: Our results suggest that μ receptor-D receptor heterodimers may be involved in the dopamine-independent expression of locomotor sensitization to opiates.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/bph.13908DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5554314PMC
September 2017

κ Opioid receptor activation in different brain regions differentially modulates anxiety-related behaviors in mice.

Neuropharmacology 2016 11 20;110(Pt A):92-101. Epub 2016 Apr 20.

Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica and Collaborative Innovation Center for Brain Science, Chinese Academy of Science, Shanghai 201203, China. Electronic address:

κ Opioid receptor system is widely implicated in the regulation of emotion. However, the findings about the role on anxiety in rodents are highly controversial, since both anxiogenic- and anxiolytic-like effects have been reported with κ opioid receptor activation. The mechanism and the underlying neuroanatomical substrates are unexplored. In the present study, we first investigated the effects of κ agonist U50,488H on anxiety-related behaviors over a wide range of doses, and we found that U50,488H produced dual effects in anxiety, with low dose being anxiogenic and high dose being anxiolytic. To assess the potential neuroanatomical substrates, we used phosphorylation of extracellular signal-related kinase1/2 (pERK1/2) to map the underlying neural circuits. We found that the anxiogenic effect of U50,488H was paralleled by an increase of pERK1/2 in the nucleus accumbens, whereas the anxiolytic effect was paralleled by an increase of pERK1/2 in the lateral septal nucleus. We then examined the behavioral consequences with locally microinjection of U50,488H, and we found that microinjection of U50,488H into the nucleus accumbens exerted anxiogenic-like effects, whereas microinjection of U50,488H into the lateral septal nucleus. Both effects can be abolished by κ antagonist nor-BNI pretreatment. To the best of our knowledge, the present work firstly provides the neuroanatomical sites that mediating the dual anxiogenic- and anxiolytic-like effects of U50,488H in mice. This study may help to explain current controversial role of κ receptor activation in anxiety-related behaviors in rodents, and may open new perspectives in the areas of anxiety disorders and κ receptor function.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.neuropharm.2016.04.022DOI Listing
November 2016

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

Br J Pharmacol 2015 Oct 23;172(20):4847-63. Epub 2015 Sep 23.

Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica and Collaborative Innovation Center for Brain Science, Chinese Academy of Science, Shanghai, China.

Background And Purpose: β-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 β-arrestins to the activated GPCRs is required for ERK1/2 activation. Our previous studies showed that δ receptors activate ERK1/2 through a β-arrestin-dependent mechanism without inducing β-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 β-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 β-arrestin2 was found to act downstream of the EGFR transactivation. Moreover, we demonstrated that coupling of the PKCδ/EGFR/β-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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/bph.13254DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4621978PMC
October 2015

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.

Eur J Pharmacol 2014 Oct 3;740:455-63. Epub 2014 Jul 3.

State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zu Chong Zhi Road, Shanghai 201203, China. Electronic address:

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ejphar.2014.06.045DOI Listing
October 2014

Dorsal hippocampal NMDA receptor blockade impairs extinction of naloxone-precipitated conditioned place aversion in acute morphine-treated rats by suppressing ERK and CREB phosphorylation in the basolateral amygdala.

Br J Pharmacol 2015 Jan 1;172(2):482-91. Epub 2014 Jul 1.

Department of Pharmacology, China Pharmaceutical University, Nanjing, China; State Key Laboratory of Drug Research, Key Laboratory of Receptor Research, Shanghai Institute of Materia Media, Chinese Academy of Sciences, Shanghai, China.

Background And Purpose: Substantial evidence shows that negative reinforcement resulting from the aversive affective consequences of opiate withdrawal may play a crucial role in drug relapse. Understanding the mechanisms underlying the loss (extinction) of conditioned aversion of drug withdrawal could facilitate the treatment of drug addiction.

Experimental Approach: Naloxone-induced conditioned place aversion (CPA) of Sprague-Dawley rats was used to measure conditioned aversion. An NMDA receptor antagonist and MAPK kinase inhibitor were applied through intracranial injections. The phosphorylation of ERK and cAMP response element-binding protein (CREB) was detected using Western blot.

Key Results: The extinction of CPA behaviour increased the phosphorylation of ERK and CREB in the dorsal hippocampus (DH) and basolateral amygdala (BLA), but not in the central amygdala (CeA). Intra-DH injection of AP5 or intra-BLA injection of AP-5 or U0126 before extinction training significantly attenuated ERK and CREB phosphorylation in the BLA and impaired the extinction of CPA behaviour. Although intra-DH injections of AP-5 attenuated extinction training-induced activation of the ERK-CREB pathway in the BLA, intra-BLA injection of AP5 had no effect on extinction training-induced activation of the ERK-CREB pathway in the DH.

Conclusions And Implications: These results suggest that activation of ERK and CREB in the BLA and DH is involved in the extinction of CPA behaviour and that the DH, via a direct or indirect pathway, modulates the activity of ERK and CREB in the BLA through activation of NMDA receptors after extinction training. Understanding the mechanisms underlying the extinction of conditioned aversion could facilitate the treatment of drug addiction.

Linked Articles: This article is part of a themed section on Opioids: New Pathways to Functional Selectivity. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2015.172.issue-2.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/bph.12671DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4292962PMC
January 2015

Extinction of aversive memories associated with morphine withdrawal requires ERK-mediated epigenetic regulation of brain-derived neurotrophic factor transcription in the rat ventromedial prefrontal cortex.

J Neurosci 2012 Oct;32(40):13763-75

State Key Laboratory of Drug Research, Shanghai Institute of Materia Media, Chinese Academy of Sciences, Shanghai 201203, China.

Recent evidence suggests that histone deacetylase (HDAC) inhibitors facilitate extinction of rewarding memory of drug taking. However, little is known about the role of chromatin modification in the extinction of aversive memory of drug withdrawal. In this study, we used conditioned place aversion (CPA), a highly sensitive model for measuring aversive memory of drug withdrawal, to investigate the role of epigenetic regulation of brain-derived neurotrophic factor (BDNF) gene expression in extinction of aversive memory. We found that CPA extinction training induced an increase in recruiting cAMP response element-binding protein (CREB) to and acetylation of histone H3 at the promoters of BDNF exon I transcript and increased BDNF mRNA and protein expression in the ventromedial prefrontal cortex (vmPFC) of acute morphine-dependent rats and that such epigenetic regulation of BDNF gene transcription could be facilitated or diminished by intra-vmPFC infusion of HDAC inhibitor trichostatin A or extracellular signal-regulated kinase (ERK) inhibitor U0126 (1,4-diamino-2,3-dicyano-1,4-bis(methylthio)butadiene) before extinction training. Correspondingly, disruption of the epigenetic regulation of BDNF gene transcription with U0126 or suppression of BDNF signaling with Trk receptor antagonist K252a or BDNF scavenger tyrosine kinase receptor B (TrkB)-Fc blocked extinction of CPA behavior. We also found that extinction training-induced activation of ERK and CREB and extinction of CPA behavior could be potentiated or suppressed by intra-vmPFC infusion of d-cycloserine, a NMDA receptor partial agonist or aminophosphonopentanoic acid, a NMDA receptor antagonist. We conclude that extinction of aversive memory of morphine withdrawal requires epigenetic regulation of BDNF gene transcription in the vmPFC through activation of the ERK-CREB signaling pathway perhaps in a NMDA receptor-dependent manner.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1523/JNEUROSCI.1991-12.2012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6704794PMC
October 2012

Actin polymerization-dependent increase in synaptic Arc/Arg3.1 expression in the amygdala is crucial for the expression of aversive memory associated with drug withdrawal.

J Neurosci 2012 Aug;32(35):12005-17

State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 201203, China.

Aversive memories associated with drug withdrawal may contribute to persistent drug seeking. Molecular mechanisms that are critical for aversive memory formation have yet to be elucidated. Recently, we showed in a rat conditioned place aversion (CPA) model that synaptic actin polymerization in the amygdala were required for aversive memory information. Here, we demonstrated that actin polymerization within the amygdala triggered transportation of activity-regulated cytoskeletal-associated protein (Arc/Arg3.1) into amygdalar synapses. Increased synaptic Arc/Arg3.1 expression contributed to aversive memory formation by regulating synaptic AMPA receptor (AMPAR) endocytosis, as in vivo knockdown of amygdalar Arc/Arg3.1 with Arc/Arg3.1-shRNA prevented both AMPAR endocytosis and CPA formation. We also demonstrated that conditioned morphine withdrawal led to induction of LTD in the amygdala through AMPAR endocytosis. We further demonstrated that Arc/Arg3.1-regulated AMPAR endocytosis was GluR2 dependent, as intra-amygdala injection of Tat-GluR2(3Y), a GluR2-derived peptide that has been shown to specifically block regulated, but not constitutive, AMPAR endocytosis, prevented AMPAR endocytosis, LTD induction, and aversive memory formation. Therefore, this study extends previous studies on the role of actin polymerization in synaptic plasticity and memory formation by revealing the critical molecular events involved in aversive memory formation as well as LTD induction, and by showing that Arc/Arg3.1 is a crucial mediator for actin polymerization functions, and, thus, underscores the unknown details of how actin polymerization mediates synaptic plasticity and memory.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1523/JNEUROSCI.0871-12.2012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6621511PMC
August 2012

Effects of ATPM-ET, a novel κ agonist with partial μ activity, on physical dependence and behavior sensitization in mice.

Acta Pharmacol Sin 2010 Dec 22;31(12):1547-52. Epub 2010 Nov 22.

State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 201203, China.

Aim: to investigate the effects of ATPM-ET [(-)-3-N-Ethylaminothiazolo [5,4-b]-N-cyclopropylmethylmorphinan hydrochloride] on physical dependence and behavioral sensitization to morphine in mice.

Methods: the pharmacological profile of ATPM-ET was characterized using competitive binding and GTPγS binding assays. We then examined the antinociceptive effects of ATPM-ET in the hot plate test. Morphine dependence assay and behavioral sensitization assay were used to determine the effect of ATPM-ET on physical dependence and behavior sensitization to morphine in mice.

Results: the binding assay indicated that ATPM-ET ATPM-ET exhibited a high affinity to both κ- and μ-opioid receptors with K(i) values of 0.15 nmol/L and 4.7 nmol/L, respectively, indicating it was a full κ-opioid receptor agonist and a partial μ-opioid receptor agonist. In the hot plate test, ATPM-ET produced a dose-dependent antinociceptive effect, with an ED(50) value of 2.68 (2.34-3.07) mg/kg. Administration of ATPM-ET (1 and 2 mg/kg, sc) prior to naloxone (3.0 mg/kg, sc) injection significantly inhibited withdrawal jumping of mice. In addition, ATPM-ET (1 and 2 mg/kg, sc) also showed a trend toward decreasing morphine withdrawal-induced weight loss. ATPM-ET (1.5 and 3 mg/kg, sc) 15 min before the morphine challenge significantly inhibited the morphine-induced behavior sensitization (P<0.05).

Conclusion: ATPM-ET may have potential as a therapeutic agent for the treatment of drug abuse.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/aps.2010.164DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4002947PMC
December 2010

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

J Cell Sci 2010 Dec 23;123(Pt 24):4259-70. Epub 2010 Nov 23.

State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, 201203, China.

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 β-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 β-arrestin1/2-mediated pathway. Moreover, we found that DPDPE gained the capacity to adopt the β-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 β-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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1242/jcs.073742DOI Listing
December 2010

The role of kappa-opioid receptor activation in mediating antinociception and addiction.

Acta Pharmacol Sin 2010 Sep 23;31(9):1065-70. Epub 2010 Aug 23.

Nanjing University of Chinese Medicine, China.

The kappa-opioid receptor (KOR), a member of the opioid receptor family, is widely expressed in the central nervous system and peripheral tissues. Substantial evidence has shown that activation of KOR by agonists and endogenous opioid peptides in vivo may produce a strong analgesic effect that is free from the abuse potential and the adverse side effects of mu-opioid receptor (MOR) agonists, such as morphine. In addition, activation of the KOR has also been shown to exert an inverse effect on morphine-induced adverse actions, such as tolerance, reward, and impairment of learning and memory. Therefore, the KOR has received much attention in the effort to develop alternative analgesics to MOR agonists and agents for the treatment of drug addiction. However, KOR agonists also produce several severe undesirable side effects such as dysphoria, water diuresis, salivation, emesis, and sedation in nonhuman primates, which may limit the clinical utility of KOR agonists for pain and drug abuse treatment. This article will review the role of KOR activation in mediating antinociception and addiction. The possible therapeutic application of kappa-agonists in the treatment of pain and drug addiction is also discussed.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/aps.2010.138DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4002313PMC
September 2010

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

Acta Pharmacol Sin 2010 Jul 21;31(7):784-90. Epub 2010 Jun 21.

Shanghai Institute of Materia Medica, Chinese Academy of Sciences, China.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/aps.2010.70DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4007732PMC
July 2010

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

J Neurosci 2010 Apr;30(14):5058-70

State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Shanghai Institute For Biological Sciences, Chinese Academy of Sciences, Shanghai 201203, China.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1523/JNEUROSCI.0148-10.2010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6632807PMC
April 2010

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

Acta Pharmacol Sin 2010 Apr 15;31(4):387-92. Epub 2010 Mar 15.

State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/aps.2010.16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4007664PMC
April 2010

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

Acta Pharmacol Sin 2010 Apr 15;31(4):393-8. Epub 2010 Mar 15.

School of Pharmacy, China Pharmaceutical University, Nanjing 210009, China.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/aps.2010.19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4007660PMC
April 2010

Involvement of actin rearrangements within the amygdala and the dorsal hippocampus in aversive memories of drug withdrawal in acute morphine-dependent rats.

J Neurosci 2009 Sep;29(39):12244-54

State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China.

Aversive memories of drug withdrawal can generate a motivational state leading to compulsive drug taking. Changes in synaptic plasticity may be involved in the formation of aversive memories. Dynamic rearrangement of the cytoskeletal actin, a major structural component of the dendritic spine, regulates synaptic plasticity. Here, the potential involvement of actin rearrangements in the induction of aversive memories of morphine withdrawal was examined. We found that lesions of the amygdala or dorsal hippocampus (DH) but not nucleus accumbens (NAc) impaired conditioned place aversion (CPA) of acute morphine-dependent rats. Accordingly, conditioned morphine withdrawal induced actin rearrangements in the amygdala and the DH but not in the NAc. In addition, we found that conditioned morphine withdrawal also increased activity-regulated cytoskeletal-associated protein (Arc) expression in the amygdala but not in the DH, although actin rearrangements were observed in both areas. We further found that inhibition of actin rearrangements by intra-amygdala or intra-DH injections of latrunculin A, an inhibitor of actin polymerization, significantly attenuated CPA. Furthermore, we found that manipulation of amygdala beta-adrenoceptor activity by its antagonist propranolol and agonist clenbuterol differentially altered actin rearrangements in the DH. Therefore, our findings reveal that actin rearrangements in the amygdala and the DH are required for the acquisition and consolidation of the aversive memories of drug withdrawal and that the beta-noradrenergic system within the amygdala modulates aversive memory consolidation by regulating actin rearrangements but not Arc protein expression in the DH, which is distinct from its role in modulation of inhibitory avoidance memory.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1523/JNEUROSCI.1970-09.2009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6666133PMC
September 2009

Glutamate receptors in the dorsal hippocampus mediate the acquisition, but not the expression, of conditioned place aversion induced by acute morphine withdrawal in rats.

Acta Pharmacol Sin 2009 Oct 21;30(10):1385-91. Epub 2009 Sep 21.

State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China.

Aim: To evaluate the role of glutamate receptors in the dorsal hippocampus (DH) in the motivational component of morphine withdrawal.

Methods: NMDA receptor antagonist D-AP5 (5 microg/0.5 microL per side) or AMPA receptor antagonist NBQX (2 microg/0.5 microL per side) was microinjected into DH of rats. Conditioned place aversion (CPA) induced by naloxone-precipitated morphine withdrawal were assessed.

Results: Preconditioning microinjection of D-AP5 or NBQX into the DH impaired the acquisition of CPA in acute morphine-dependent rats. However, intra-DH microinjection of D-AP5 or NBQX after conditioning but before the testing session had no effect on the expression of CPA.

Conclusion: Our results suggest that NMDA and AMPA receptors in the dorsal hippocampus are involved in the acquisition, but not in the expression, of the negative motivational components of acute morphine withdrawal in rats.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/aps.2009.130DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4007332PMC
October 2009

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

FEBS J 2009 Apr;276(7):2022-36

State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/j.1742-4658.2009.06938.xDOI Listing
April 2009

Expression pattern of neural synaptic plasticity marker-Arc in different brain regions induced by conditioned drug withdrawal from acute morphine-dependent rats.

Acta Pharmacol Sin 2009 Mar;30(3):282-90

State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China.

Aim: The immediate early gene Arc (activity-regulated cytoskeletal-associated protein) mRNA and protein are induced by strong synaptic activation and rapidly transported into dendrites, where they localize at active synaptic sites. Thus, the Arc mRNA and protein are proposed as a marker of neuronal reactivity to map the neural substrates that are recruited by various stimuli. In the present study, we examined the expression of Arc protein induced by conditioned naloxone-precipitated drug withdrawal in different brain regions of acute morphine-dependent rats. The objective of the present study was to address the specific neural circuits involved in conditioned place aversion (CPA) that has not yet been well characterized.

Methods: Place aversion was elicited by conditioned naloxone-precipitated drug withdrawal following exposure to a single dose of morphine. An immunohistochemical method was employed to detect the expression of Arc, which was used as a plasticity marker to trace the brain areas that contribute to the formation of the place aversion.

Results: Marked increases in Arc protein levels were found in the medial and lateral prefrontal cortex, the sensory cortex, the lateral striatum and the amygdala. This effect was more pronounced in the basolateral amygdala (BLA), the central nucleus of the amygdala (CeA), and the bed nucleus of the striatal terminals (BNST) when compared with the control group.

Conclusion: Our results suggest that these brain regions may play key roles in mediating the negative motivational component of opiate withdrawal.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/aps.2009.10DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4002397PMC
March 2009

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.

J Pharmacol Exp Ther 2009 Apr 9;329(1):306-13. Epub 2009 Jan 9.

School of Life Science and Biopharmaceutics, Shenyang Pharmaceutical University, Shenyang, China.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1124/jpet.108.142802DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2670601PMC
April 2009

Role of Src in ligand-specific regulation of delta-opioid receptor desensitization and internalization.

J Neurochem 2009 Jan 10;108(1):102-14. Epub 2008 Nov 10.

State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China.

The opioid receptors are a member of G protein-coupled receptors that mediate physiological effects of endogenous opioid peptides and structurally distinct opioid alkaloids. Although it is well characterized that there is differential receptor desensitization and internalization properties following activation by distinct agonists, the underlying mechanisms remain elusive. We investigated the signaling events of delta-opioid receptor (deltaOR) initiated by two ligands, DPDPE and TIPP. We found that although both ligands inhibited adenylyl cyclase (AC) and activated ERK1/2, only DPDPE induced desensitization and internalization of the deltaOR. We further found that DPDPE, instead of TIPP, could activate GRK2 by phosphorylating the non-receptor tyrosine kinase Src and translocating it to membrane receptors. Activation of GRK2 led to the phosphorylation of serine residues in the C-terminal tail, which facilitates beta-arrestin1/2 membrane translocation. Meanwhile, we also found that DPDPE promoted beta-arrestin1 dephosphorylation in a Src-dependent manner. Thus, DPDPE appears to strengthen beta-arrestin function by dual regulations: promoting beta-arrestin recruitment and increasing beta-arrestin dephosphorylation at the plasma membrane in a Src-dependent manner. All effects initiated by DPDPE could be abolished or suppressed by PP2, an inhibitor of Src. Morphine, which has been previously shown to be unable to desensitize or internalize deltaOR, also behaved as TIPP in failure to utilize Src to regulate deltaOR signaling. These findings point to the existence of agonist-specific utilization of Src to regulate deltaOR signaling and reveal the molecular events by which Src modulates deltaOR responsiveness.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/j.1471-4159.2008.05740.xDOI Listing
January 2009

LPK-26, a novel kappa-opioid receptor agonist with potent antinociceptive effects and low dependence potential.

Eur J Pharmacol 2008 Apr 19;584(2-3):306-11. Epub 2008 Feb 19.

State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 201203, People's Republic of China.

Analgesics such as morphine cause many side effects including addiction, but kappa-opioid receptor agonist can produce antinociception without morphine-like side effects. With the aim of developing new and potent analgesics with lower abuse potential, we studied the antinociceptive and physical dependent properties of a derivate of ICI-199441, an analogue of (-)U50,488H, named (2-(3,4-dichloro)-phenyl)-N-methyl-N-[(1S)-1-(2-isopropyl)-2-(1-(3-pyrrolinyl))ethyl] acetamides (LPK-26). LPK-26 showed a high affinity to kappa-opioid receptor with the Ki value of 0.64 nM and the low affinities to micro-opioid receptor and delta-opioid receptor with the Ki values of 1170 nM and >10,000 nM, respectively. It stimulated [(35)S]GTPgammaS binding to G-proteins with an EC50 value of 0.0094 nM. In vivo, LPK-26 was more potent than (-)U50,488H and morphine in analgesia, with the ED50 values of 0.049 mg/kg and 0.0084 mg/kg in hot plat and acetic acid writhing tests, respectively. Moreover, LPK-26 failed to induce physical dependence, but it could suppress naloxone-precipitated jumping in mice when given simultaneously with morphine. Taken together, our results show that LPK-26 is a novel selective kappa-opioid receptor agonist with highly potent antinociception effects and low physical dependence potential. It may be valuable for the development of analgesic and drug that can be used to reduce morphine-induced physical dependence.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ejphar.2008.02.028DOI Listing
April 2008

Expression changes of hippocampal energy metabolism enzymes contribute to behavioural abnormalities during chronic morphine treatment.

Cell Res 2007 Aug;17(8):689-700

State key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China.

Dependence and impairment of learning and memory are two well-established features caused by abused drugs such as opioids. The hippocampus is an important region associated with both drug dependence and learning and memory. However, the molecular events in hippocampus following exposure to abused drugs such as opioids are not well understood. Here we examined the effect of chronic morphine treatment on hippocampal protein expression by proteomic analyses. We found that chronic exposure of mice to morphine for 10 days produced robust morphine withdrawal jumping and memory impairment, and also resulted in a significant downregulation of hippocampal protein levels of three metabolic enzymes, including Fe-S protein 1 of NADH dehydrogenase, dihydrolipoamide acetyltransferase or E2 component of the pyruvate dehydrogenase complex and lactate dehydrogenase 2. Further real-time quantitative PCR analyses confirmed that the levels of the corresponding mRNAs were also remarkably reduced. Consistent with these findings, lower ATP levels and an impaired ability to convert glucose into ATP were also observed in the hippocampus of chronically treated mice. Opioid antagonist naltrexone administrated concomitantly with morphine significantly suppressed morphine withdrawal jumping and reversed the downregulation of these proteins. Acute exposure to morphine also produced robust morphine withdrawal jumping and significant memory impairment, but failed to decrease the expression of these three proteins. Intrahippocampal injection of D-glucose before morphine administration significantly enhanced ATP levels and suppressed morphine withdrawal jumping and memory impairment in acute morphine-treated but not in chronic morphine-treated mice. Intraperitoneal injection of high dose of D-glucose shows a similar effect on morphine-induced withdrawal jumping as the central treatment. Taken together, our results suggest that reduced expression of the three metabolic enzymes in the hippocampus as a result of chronic morphine treatment contributes to the development of drug-induced symptoms such as morphine withdrawal jumping and memory impairment.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/cr.2007.63DOI Listing
August 2007

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

Biochem J 2007 Sep;406(2):215-21

State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1042/BJ20070186DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1948956PMC
September 2007

Involvement of dopamine system in regulation of Na+,K+-ATPase in the striatum upon activation of opioid receptors by morphine.

Mol Pharmacol 2007 Feb 26;71(2):519-30. Epub 2006 Oct 26.

State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zuchongzhi Rd., Shanghai 201203, China.

The striatum is believed to be a crucial brain region associated with drug reward. Adaptive alteration of neurochemistry in this area might be one potential mechanism underlying drug dependence. It has been proposed that the dysfunction of Na+,K+-ATPase function is involved in morphine tolerance and dependence. The present study, therefore, was undertaken to study the adaptation of the striatal Na+,K+-ATPase activity in response to morphine treatment. The results demonstrated that in vivo short-term morphine treatment stimulated Na+,K+-ATPase activity in a dose-dependent manner. This action could be significantly inhibited by D2-like dopamine receptor antagonist S(-)-3-chloro-5-ethyl-N-[(1-ethyl-2-pyrrolidinyl)methyl]-6-hydroxy-2-methoxybenzamine (eticlopride). Contrary to shortterm morphine treatment, long-term morphine treatment significantly suppressed Na+,K+-ATPase activity. This effect could be significantly inhibited by D(1)-like dopamine receptor antagonist R(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrochloride (SCH 23390). However, both short-term and long-term morphine treatment-induced changes in Na+,K+-ATPase activity could be reversed by opioid receptor antagonist naltrexone. It was further found that cAMP-dependent protein kinase (PKA) was crucially involved in regulating Na+,K+-ATPase activity by morphine. Different regulation of the phosphorylation levels of the alpha3 subunit of Na+,K+-ATPase by PKA was related to the distinct modulations of Na+,K+-ATPase by short-term and long-term morphine treatment. Short-term morphine treatment inhibited PKA activity and then decreased the phosphorylation of Na+,K+-ATPase, leading to increase in enzyme activity. These effects were sensitive to eticlopride or naltrexone. Conversely, long-term morphine treatment stimulated PKA activity and then increased the phosphorylation of Na+,K+-ATPase, leading to the reduction of enzyme activity. These effects were sensitive to SCH 23390 or naltrexone. These findings demonstrate that dopamine receptors are involved in regulation of Na+,K+-ATPase activity after activation of opioid receptors by morphine.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1124/mol.106.029561DOI Listing
February 2007

Involvement of cAMP/cAMP-dependent protein kinase signaling pathway in regulation of Na+,K+-ATPase upon activation of opioid receptors by morphine.

Mol Pharmacol 2006 Mar 29;69(3):866-76. Epub 2005 Nov 29.

State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, No.555 Zuchongzhi Rd., Shanghai 201203, China.

The depolarization of neurons induced by impairment of Na+,K+-ATPase activity after long-term opiate treatment has been shown to involve the development of opioid dependence. However, the mechanisms underlying changes in Na+,K+-ATPase activity after opioid treatment are unclear. The best-established molecular adaptation to long-term opioid exposure is up-regulation of the cAMP/cAMP-dependent protein kinase (PKA) signaling pathway; this study, therefore, was undertaken to investigate the role of up-regulation of cAMP/PKA signaling pathway in alteration of the mouse hippocampal Na+,K+-ATPase activity. The results demonstrated that short-term morphine treatment dose dependently stimulated Na+,K+-ATPase activity. This action could be significantly suppressed by adenylyl cyclase activator 7beta-acetoxy-8,13-epoxy-1alpha,6beta,9alpha-trihydroxylabd-14-en-11-one (forskolin), or the cAMP analog dibutyryl-cAMP. Contrary to short-term morphine treatment, long-term treatment significantly inhibited Na+,K+-ATPase activity. Moreover, an additional decrease in Na+,K+-ATPase activity was observed by naloxone precipitation. The effects of both short- and long-term morphine treatment on Na+,K+-ATPase activity were naltrexone-reversible. The regulation of Na+,K+-ATPase activity by morphine was inversely correlated with intracellular cAMP accumulation. N-[2-(4-Bromocinnamylamino)ethyl]-5-isoquinoline (H89), a specific PKA inhibitor, mimicked the stimulatory effect of short-term morphine but antagonized the inhibitory effect of long-term morphine treatment on Na+,K+-ATPase activity. However, okadaic acid, a protein phosphatase inhibitor, suppressed short-term morphine stimulation but potentiated long-term morphine inhibition of Na+,K+-ATPase activity. The regulation of Na+,K+-ATPase activity by morphine treatment seemed to associate with the alteration in phosphorylation level but not to be relevant to the change in abundance of Na+,K+-ATPase. These findings strongly demonstrate that cAMP/PKA signaling pathway involves regulation of Na+,K+-ATPase activity after activation of opioid receptors.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1124/mol.105.016501DOI Listing
March 2006

Effect of chronic treatment of ohmefentanyl stereoisomers on cyclic AMP formation in Sf9 insect cells expressing human mu-opioid receptors.

Life Sci 2004 Apr;74(24):3001-8

2nd Department of Pharmacology, Shanghai Institute of Materia Medica, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 201203, China.

The binding affinity of ohmefentanyl stereoisomers for mu-opioid receptors and the effect of chronic ohmefentanyl stereoisomers pretreatments on intracellular cAMP formation were investigated in Sf9 insect cells expressing human mu-opioid receptors (Sf9-mu cells). Competitive assay of [3H]ohmefentanyl binding revealed that these isomers had high affinity for micro-opioid receptors in Sf9-mu cells. Isomer F9204 had the highest affinity for mu-opioid receptors with the Ki value of 1.66 +/- 0.28 nM. After pretreated Sf9-mu cells with increasing concentrations of these isomers for 6 h, addition of naloxone (1 microM) precipitated an overshoot of foskolin-stimulated cAMP accumulation. The ability of these isomers to induce cAMP overshoot differed greatly with the order of F9202>F9205>F9208>F9206>F9204>F9207. Of these isomers, F9202 was 2.7-fold less potent than F9204 in receptor binding affinity, but 71.5-fold more potent in ability to induce cAMP overshoot. These results suggested that there was a significant stereo-structural difference among ohmefentanyl stereoisomers in ability to induce naloxone-precipitated cAMP overshoot in Sf9-mu cells.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.lfs.2003.10.027DOI Listing
April 2004

Effects of ohmefentanyl stereoisomers on phosphorylation of cAMP- response element binding protein in cultured rat hippocampal neurons.

Acta Pharmacol Sin 2003 Dec;24(12):1253-8

Shanghai Institute of Materia Medica, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China.

Aim: To define the effects and signal pathways of ohmefentanyl stereoisomers [(-)-cis-(3R,4S,2'R) OMF (F9202), (+)-cis-(3R,4S,2'S) OMF (F9204), and (-)-cis-(3S,4S,2'R) OMF (F9203)] on the phosphorylation of cAMP-response element binding protein (CREB) in cultured rat hippocampal neurons.

Methods: The effects of the three OMF stereoisomers and morphine (Mor) on cAMP accumulation and CREB phosphorylation were monitored by radioimmunoassay and Western blot analysis, respectively.

Results: The three OMF stereoisomers and Mor could all partially inhibit forskolin-stimulated (25 micromol/L, 15 min) cAMP accumulation in a dose-dependent manner and this effect could be reversed by naloxone. F9202, F9204, and Mor could significantly increase CREB phosphorylation from 2.88 to 3.59 folds over control levels after 30-min exposure. This effect was reversed by naloxone, but F9203 failed to increase CREB phosphorylation. KN-62 and staurosporine significantly blocked the opioids- induced CREB phosphorylation, while H-89 and PD 98059 had no effect on the actions.

Conclusion: Mor, F9202, and F9204, which could induce psychological dependence affected via the micro-opioid receptor, stimulated intracellular signal pathways involving Ca2+/calmodulin-dependent protein kinases (CCDPK) and protein kinase C (PKC) pathways, which in turn initiated CREB phosphorylation. F9203, which could not induce dependence, had no effect on CREB phosphorylation in hippocampal neurons. The increased CREB phosphorylation in hippocampal neurons may play a role in opioids dependence.
View Article and Find Full Text PDF

Download full-text PDF

Source
December 2003

Binding affinity to and dependence on some opioids in Sf9 insect cells expressing human mu-opioid receptor.

Acta Pharmacol Sin 2003 Sep;24(9):859-63

Shanghai Institute of Materia Medica, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 201203.

Aim: To investigate the receptor binding affinity and naloxone-precipitated cAMP overshoot of dihydroetorphine, fentanyl, heroin, and pethidine in Sf9 insect cells expressing human mu-opioid receptor (Sf9-mu cells).

Methods: Competitive binding assay of [3H]ohmefentanyl was used to reveal the affinity for mu-opioid receptor in Sf9-mu cells. [3H]cAMP RIA was used to determine cAMP level. Antinociceptive activity was evaluated using degree 55 mouse hot plate test. Naloxone-precipitated withdrawal jumping was used to reflect physical dependence in mice.

Results: All drugs displayed antinociceptive activity and produced physical dependence in mice. The K(i) values of dihydroetorphine, fentanyl, heroin, and pethidine in competitive binding assay were (0.85+/-0.20) nmol, (59.1+/-11.7) nmol, (0.36+/-0.13) micromol, and (12.2+/-3.8) micromol respectively. The binding affinities of these drugs for mu-opioid receptor in Sf9-mu cells were paralleled to their antinociceptive activities in mice. After chronic pretreatment with these drugs, naloxone induced cAMP withdrawal overshoot in Sf9-mu cells. The dependence index in Sf9-mu cells was calculated as K(i) value in competitive binding assay over EC(50) value in naloxone-precipitated cAMP assay. The physical dependence index in mice was calculated as antinociceptive ED(50)/withdrawal jumping cumulative ED(50). There was a good linear correlation between dependence index in Sf9-mu cells and physical dependence index in mice.

Conclusion: The Sf9-mu cells could be used as a cell model to evaluate the receptor binding affinity and physical dependent liability of analgesic agents.
View Article and Find Full Text PDF

Download full-text PDF

Source
September 2003

Opioid activity of C8813, a novel and potent opioid analgesic.

Life Sci 2003 May;73(2):233-41

2nd Department of Pharmacology, Shanghai Institute of Materia Medica, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, China.

Compound trans-4-(p-bromophenyl)-4-(dimethylamino)-1-(2-thiophen-2-yl-ethyl)-cyclohexanol (C8813), structurally unrelated to morphine, is a novel analgesic. The present study examined the antinociception, opioid receptor selectivity and in vitro activity of C8813. The antinociceptive activity was evaluated using mouse hot plate and acetic acid writhing tests. In mouse hot plate test, the antinociceptive ED(50) of C8813 was 11.5 microg/kg, being 591 times and 3.4 times more potent than morphine and fentanyl respectively. In mouse writhing test, the antinociceptive ED(50) of C8813 was 16.9 microg/kg, being 55 times and 2.3 times more active than morphine and fentanyl respectively. In the opioid receptor binding assay, C8813 showed high affinity for mu-opioid receptor (K(i) = 1.37 nM) and delta-opioid receptor (K(i) = 3.24 nM) but almost no affinity for kappa-opioid receptor (at 1 microM). In the bioassay, the inhibitory effect of C8813 in the guinea-pig ileum (GPI) was 16.5 times more potent than in the mouse vas deferens (MVD). The inhibitory effects of C8813 in the GPI and MVD could be antagonized by mu-opioid receptor antagonist naloxone and delta-opioid receptor antagonist ICI174,864 respectively. However, the inhibitory effect of C8813 in the rabbit vas deferens was very weak. These results indicated that C8813 was a potent analgesic and a high affinity agonist for the mu- and delta-opioid receptors.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/s0024-3205(03)00263-7DOI Listing
May 2003

Internalization and recycling of human mu opioid receptors expressed in Sf9 insect cells.

Life Sci 2003 May;73(1):115-28

Shanghai Institute of Materia Medica, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 294 Taiyuan Road, Shanghai 200031, China.

Internalization and recycling of G protein-coupled receptors (GPCRs), such as the mu-opioid receptor, largely depend on agonist stimulation. Agonist-promoted internalization of some GPCRs has been shown to mediate receptor desensitization, resensitization, and down-regulation. In this study, we investigated whether different mu opioid agonists displayed different effects in receptor internalization and recycling, the potential mechanisms involved in ohmefentanyl-induced internalization process. In transfected Sf9 insect cells expressing 6His-tagged wild type mu opioid receptor, exposure to 100 nM ohmefentanyl caused a maximum internalization of the receptor at 30 min and receptors seemed to reappear at the cell membrane after 60 min as determined by radioligand binding assay. Ohmefentanyl-induced human mu opioid receptor internalization was concentration-dependent, with about 40% of the receptors internalized following a 30-min exposure to 1 microM ohmefentanyl. 10 microM morphine and 1 microM DAMGO could also induce about 40% internalization. The antagonist naloxone and pretreatment with pertussis toxin both blocked ohmefentanyl-induced internalization without affecting internalization themselves. Incubation with sucrose 0.45 M significantly inhibited ohmefentanyl-induced internalization of the mu receptor. The removal of agonists ohmefentanyl and morphine resulted in the receptors gradually returning to the cell surface over a 60 min period, while the removal of agonist DAMGO only partly resulted in the receptor recycling. The results of this study suggest that ohmefentanyl-induced internalization of human mu opioid receptor in Sf9 insect cells occurs via Gi/o protein-dependent process that likely involves clathrin-coated pits. In addition, the recycling process displays the differential modes of action of different agonists.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/s0024-3205(03)00250-9DOI Listing
May 2003