RESUMEN
High-quality counterfeit stamp impressions made by inkjet printers remain challenging in questioned document examination and forensic analyses. A dataset comprised of various printed stamp impressions, using ten options of conditions and materials, and hand stamped impressions was generated. In this paper, we report printed impressions in pure color and high-quality printing mode are very similar to hand stamped impressions in terms of their microscopic characteristics. These similarities may lead to incorrect conclusions via traditional identification methods. Here, we proposed a method for identifying counterfeit stamp impressions via texture features and image quality parameters extracted from impressions. First, the statistical analysis methods were used to verify a significant difference between the printed and hand stamped impressions. Principal component analysis (PCA) was used to show the variation between the impressions, and the differences between printed and hand stamped impressions were obvious in the three-dimensional plot. After filtering the background of the stamp impressions, image processing analysis was introduced to extract features of gray level co-occurrence matrix (GLCM), segmentation-based fractal texture analysis (SFTA), local binary pattern (LBP), and image quality metrics (IQM), which were used to characterize the stamp impressions. Finally, specific cases were simulated by random selection, based on the dataset of stamp impressions, and an evaluation system for stamp evidence was established to calculate the likelihood ratios (LRs) under two alternative hypotheses. The likelihood ratio interprets calibrated evaluations on the strength of stamp impressions as evidence. We can also balance these LRs against the rates of misleading evidence with a reasonable performance (equal error rate = 0.048). This paper provides a system to differentiate high-quality printed and hand stamped impressions with reasonable performance.
RESUMEN
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.
Asunto(s)
Analgésicos Opioides/farmacología , Benzazepinas/farmacología , Antagonistas de Dopamina/farmacología , Receptores de Dopamina D1/metabolismo , Receptores Opioides mu/metabolismo , Animales , Células Cultivadas , AMP Cíclico/metabolismo , Dopamina , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Células HEK293 , Humanos , Ratones Noqueados , Actividad Motora/efectos de los fármacos , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Proteínas Proto-Oncogénicas c-fos/metabolismo , Ratas Sprague-Dawley , Receptores de Dopamina D1/genéticaRESUMEN
AIM: To characterize the pharmacological profiles of a novel κ-opioid receptor agonist MB-1C-OH. METHODS: [(3)H]diprenorphine binding and [(35)S]GTPγS binding assays were performed to determine the agonistic properties of MB-1C-OH. Hot plate, tail flick, acetic acid-induced writhing, and formalin tests were conducted in mice to evaluate the antinociceptive actions. Forced swimming and rotarod tests of mice were used to assess the sedation and depression actions. RESULTS: In [(3)H]diprenorphine binding assay, MB-1C-OH did not bind to µ- and δ-opioid receptors at the concentration of 100 µmol/L, but showed a high affinity for κ-opioid receptor (Ki=35 nmol/L). In [(35)S]GTPγS binding assay, the compound had an Emax of 98% and an EC50 of 16.7 nmol/L for κ-opioid receptor. Subcutaneous injection of MB-1C-OH had no effects in both hot plate and tail flick tests, but produced potent antinociception in the acetic acid-induced writhing test (ED50=0.39 mg/kg), which was antagonized by pretreatment with a selective κ-opioid receptor antagonist Nor-BNI. In the formalin test, subcutaneous injection of MB-1C-OH did not affect the flinching behavior in the first phase, but significantly inhibited that in the second phase (ED50=0.87 mg/kg). In addition, the sedation or depression actions of MB-1C-OH were about 3-fold weaker than those of the classical κ agonist (-)U50,488H. CONCLUSION: MB-1C-OH is a novel κ-opioid receptor agonist that produces potent antinociception causing less sedation and depression.
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Analgésicos Opioides/farmacología , Conducta Animal/efectos de los fármacos , Isoquinolinas/farmacología , Umbral del Dolor/efectos de los fármacos , Dolor/prevención & control , Receptores Opioides kappa/agonistas , Vigilia/efectos de los fármacos , Analgésicos Opioides/metabolismo , Analgésicos Opioides/toxicidad , Animales , Unión Competitiva , Células CHO , Cricetulus , Depresión/inducido químicamente , Depresión/metabolismo , Depresión/psicología , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Humanos , Isoquinolinas/metabolismo , Ligandos , Masculino , Ratones , Actividad Motora/efectos de los fármacos , Dolor/metabolismo , Dolor/fisiopatología , Dolor/psicología , Unión Proteica , Ratas , Receptores Opioides kappa/genética , Receptores Opioides kappa/metabolismo , TransfecciónRESUMEN
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.
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Dependencia de Heroína/tratamiento farmacológico , Morfinanos/uso terapéutico , Antagonistas de Narcóticos/uso terapéutico , Receptores Opioides kappa/agonistas , Receptores Opioides mu/agonistas , Receptores Opioides mu/antagonistas & inhibidores , Analgésicos Opioides/administración & dosificación , Animales , Condicionamiento Psicológico , Comportamiento de Búsqueda de Drogas/efectos de los fármacos , Masculino , Ratones , Morfina/administración & dosificación , Dolor/tratamiento farmacológico , Ratas Sprague-Dawley , AutoadministraciónRESUMEN
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.
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Conducta Animal/efectos de los fármacos , Morfinanos/farmacología , Dependencia de Morfina/tratamiento farmacológico , Morfina/farmacología , Narcóticos/farmacología , Receptores Opioides kappa/agonistas , Receptores Opioides mu/agonistas , Animales , Relación Dosis-Respuesta a Droga , Calor , Masculino , Ratones , Dependencia de Morfina/metabolismo , Dependencia de Morfina/psicología , Actividad Motora/efectos de los fármacos , Naloxona/farmacología , Antagonistas de Narcóticos/farmacología , Dimensión del Dolor , Receptores Opioides kappa/metabolismo , Receptores Opioides mu/metabolismoRESUMEN
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.
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Analgésicos/uso terapéutico , Receptores Opioides kappa/agonistas , Receptores Opioides kappa/metabolismo , Trastornos Relacionados con Sustancias/tratamiento farmacológico , Analgésicos/farmacología , Animales , Humanos , Dolor/tratamiento farmacológicoRESUMEN
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.
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Agonistas del Receptor de Adenosina A1 , Adenosina/análogos & derivados , Receptores Opioides delta/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Adenosina/farmacología , Animales , Células CHO , Cricetinae , Cricetulus , AMP Cíclico/metabolismo , Semivida , Humanos , Fosforilación/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Ensayo de Unión Radioligante , Receptores Opioides delta/metabolismo , Factores de TiempoRESUMEN
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.
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Analgésicos Opioides/farmacología , Etorfina/análogos & derivados , Fentanilo/análogos & derivados , Morfina/farmacología , Receptores Opioides mu/metabolismo , Animales , Células CHO , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Colforsina/metabolismo , Cricetinae , Cricetulus , AMP Cíclico/metabolismo , Endocitosis/efectos de los fármacos , Etorfina/farmacología , Fentanilo/farmacología , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Trastornos Relacionados con Opioides/metabolismo , Receptores Opioides mu/agonistasRESUMEN
Unexpected substituent on the well-known morphine skeleton is described to be account for highly selective and potent mu opioid ligands, which is strongly connected to substituted aromatic groups on this omitted 8alpha-position.
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Ligandos , Morfina/química , Receptores Opioides mu/metabolismo , Cristalografía por Rayos X , Humanos , Conformación Molecular , Unión Proteica , Tebaína/químicaRESUMEN
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.
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Analgésicos Opioides/antagonistas & inhibidores , Analgésicos Opioides/farmacología , Dependencia de Heroína/tratamiento farmacológico , Dependencia de Heroína/psicología , Morfinanos/farmacología , Morfina/antagonistas & inhibidores , Morfina/farmacología , Receptores Opioides kappa/agonistas , Receptores Opioides mu/agonistas , Receptores Opioides mu/antagonistas & inhibidores , 3,4-Dicloro-N-metil-N-(2-(1-pirrolidinil)-ciclohexil)-bencenacetamida, (trans)-Isómero/farmacología , Músculos Abdominales/efectos de los fármacos , Analgésicos no Narcóticos/farmacología , Animales , Relación Dosis-Respuesta a Droga , Tolerancia a Medicamentos , Calor , Masculino , Ratones , Dimensión del Dolor/efectos de los fármacos , Equilibrio Postural/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Tiempo de Reacción/efectos de los fármacos , Receptores Opioides delta/efectos de los fármacos , AutoadministraciónRESUMEN
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.
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Receptores Opioides delta/metabolismo , Transducción de Señal/fisiología , Familia-src Quinasas/fisiología , Adenilil Ciclasas/metabolismo , Analgésicos Opioides/farmacología , Animales , Arrestinas/metabolismo , Células CHO , Cricetinae , Cricetulus , Relación Dosis-Respuesta a Droga , Encefalina D-Penicilamina (2,5)/farmacología , Citometría de Flujo , Quinasa 2 del Receptor Acoplado a Proteína-G/metabolismo , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Morfina/farmacología , Oligopéptidos/farmacología , Unión Proteica/efectos de los fármacos , Transporte de Proteínas/efectos de los fármacos , Serina/metabolismo , Transducción de Señal/efectos de los fármacos , Tetrahidroisoquinolinas/farmacología , Transfección , beta-ArrestinasRESUMEN
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.
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Analgésicos Opioides/farmacología , Trastornos Relacionados con Opioides/prevención & control , Umbral del Dolor/efectos de los fármacos , Dolor/prevención & control , Pirroles/farmacología , Receptores Opioides kappa/agonistas , Ácido Acético , Animales , Conducta Animal/efectos de los fármacos , Células CHO , Cricetinae , Cricetulus , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Calor/efectos adversos , Humanos , Masculino , Ratones , Actividad Motora/efectos de los fármacos , Naloxona/farmacología , Antagonistas de Narcóticos/farmacología , Dolor/etiología , Dolor/fisiopatología , Dimensión del Dolor , Unión Proteica , Ratas , Receptores Opioides delta/agonistas , Receptores Opioides delta/metabolismo , Receptores Opioides kappa/genética , Receptores Opioides kappa/metabolismo , Receptores Opioides mu/agonistas , Receptores Opioides mu/metabolismo , Factores de Tiempo , TransfecciónRESUMEN
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.