The Role Of Phosphatidylethanolamine-binding Protein In Opioid Dependence

Substance dependence also known as drug addiction is a chronic relapsing brain disorders. Opioid abuse account for a large proportion in total drug abuse in China, so opioid dependence has threatened the health of people and the stability of society and economy severely. However, the mechanisms of opioid dependence are so complicated that no effective medication is available to treat the opioid addicts. In general, it is repeated opioids exposure-induced plasticity of nervous system that results in opioid dependence, such as the upregulated threshold of brain reward system, the abnormality of motivation system, and the disturbance of study and memory induced by opioids. Opioid dependence involves the adaptation of signal transduction in neural cells of different brain region, such as upregulated adenylyl cyclase (AC) activity in the neurons of locus coeruleus. Detecting and studying these changed molecules during the development of adaptation are very important to explain the mechanisms of opioid dependence and find new therapeutic methods and targets.Phosphatidylethanolamine-binding protein (PEBP) is such a molecular whose expression was upregulated in rat hippocampus during the development of opioid dependence by proteomics analysis. PEBP is a multiple functional protein, including (1) PEPB binds on Raf-1 kinase, with possibility to inhibit ERK; (2) phosphorylated PEBP binds on G protein-coupled receptor kinase 2 (GRK2), with possibility to inhibit G protein-coupled receptor (GPCR) phosphorylation; (3) PEBP stimulates Gi activity, with possibility to affect GPCR function; (4) PEBP is the precursor of hippocampal cholinergic neurostimulating peptide (HCNP), with possibility to modulate hippopotamus cholinergic system. In summary, functions 1-3 are highly related to the regulation of opioid receptor pathway; function 4 is highly related to the regulation of nervous function during the development of opioid dependence. So, PEBP may contributes to opiod dependence through two mechanisms, one is regulating opioid receptor related signal pathway; the other is affecting nervous cholinergic system.There is no report on the issue of PEBP involvement in opioid dependence besides ours. Our previous works showed that chronic high dose of morphine treatment induced a PEBP upregulation in hippopotamus, and the signs of morphine dependence were aggregated when PEBP in hippopotamus is suppressed by antisense nucleic acids, indicating that PEBP is involved in opioid dependence. Further studies suggested that there was correlation between the activity of choline acetyl transferase and the expression of PEBP in hippocampus, indicating that PEBP may contribute to opioid dependence by regulating hippopotamus cholinergic system. As a result, the purpose of this paper is to further study if PEBP contributes to opioid dependence through regulating signal transduction on the basis of our previous achievements.In order to answer this question, three parts of experiment were designed: (1) to study the expression of PEBP and the activity of ERK in addiction-related brain regions (prefrontal cortex, hippopotamus, nucleus accumbens and striatum) during different pahses of morphine-induced conditioned place preference (CPP) of rats; (2) to study the relation between the activity of PEBP and ERK inμopioid receptor (MOR)-mediated ERK activation in cellular level; (3) to study the effects of PEBP overexpression on the functions of MOR in cellular level.Result 1, the change of PEBP expression and ERK activity in the CPP model of rat. PEBP expression level did not change significantly compared with control in prefrontal cortex, hippocampus, striatum, and nucleus accumbens of rats during CPP-mimiced three phases of morphine psychological dependence, including morphine-induced (10mg/kg) CPP expression, natural withdrawl of CPP, and low dose (2.5mg/kg) of morphine-induced reinstatement of CPP. However, the activity of ERK was found to increase notablely (about 1.5 folds of control) in prefrontal cortex after the expression of CPP, and decrease remarkably (about 0.5 fold of control) in hippocampus. No significant change of ERK activity was observed in other brain regions and phases of CPP. These results suggested that the activity of ERK is involved in the mechanisms of morphine addiction, in which the activity of ERK is modulated discriminatively in different brain regions, indicating that formation and retrieve of associated memory between drug effects and environment may involve different neural circuits. However, PEBP is excluded in this mechanism of memory-related ERK regulation. In order to interpret the role of PEBP in MOR-mediated ERK regualtion, the second part of research was carried out.Result 2, the relation between PEBP and MOR-mediated ERK regulation. In both CHO cells that highly expressing MOR and SH-SY5Y cells that endogenously expressing MOR, morphine and DAMGO all could induce a concentration- and time-dependent ERK activation, but no change of PEBP phosphorylation was observed during the opioid treatment. Chronic morhine (10μM, 36h) treatment caused no change of PEBP expression and the ERK activity, however, when the cells were precipitated with naloxone, a significant decrease of ERK activity was observed, but not PEBP phosphorylation. In SH-SY5Y cells, PKC activator PMA induced a significant enhancement of ERK and PEBP phosphorylation, but their time-effect curve was different: the former was rapid and strong (3-4 folds of control at 10min of treatment, and sustained above 1h), while the latter was slow and week (significantly only at 1h of treatment). PKC inhibitor G?6983 did not inhibit DAMGO or morphine-induced ERK activation; AC activator forskolin induced significant ERK activation but not PEBP. These results indicated that there was no correlation between MOR-mediated ERK regualtion and PEBP phosphorylation; PEBP did not regulated by cAMP/PKA pathway; PEBP was the substrate of PKC, but had nothing to do with PKC-induced rapid ERK activation. Then we further studied the relation between PEBP phosphorylation and ERK activation induced by other types of GPCR. In HEK293 cells that stably expressing Gs-coupled dopamine D1 receptors, dopamine caused a remarkable activation of ERK but not PEBP. In HEK293 cells that stably expressing Gq-coupled adrenergicα1A receptors, however, norepinephrine caused a remarkable activation of ERK, as well as PEBP, whose patter was similar to that induced by PMA. Furthermore, G?6983 completely inhibited norepinephrine-induced ERK and PEBP activation. These results suggested that it is the activation of PKC mediated byα1A receptor that results in the activation of PEBP, the mechnism of which is identical to direct stimulation of PKC. In this part, we did not find PEBP was regualted by MOR activation, so would PEBP overexpression affect MOR function? the third part of research was carried out.Result 3, effect of PEBP overexpression on MOR function. A cell line that overexpresses PEBP stably was setup on the basis of CHO-μcell firstly. Then we found that overexpression of PEBP did not affect cell cycle, basal ERK activity, MOR expression and affinity, and agonism activity of DAMGO against MOR, indicating that PEBP overexpression had no effect on basal properties of cells and MOR. Further results showed that acute treatment of DAMGO induced significant rapid MOR desensitization and decrease of membrane receptors, but PEBP overexpression did not affect these effets, which is probably duo to PEBP's no effect on DAMGO-induced MOR phosphorylation. The regulation of AC and the crosstalk with ERK are important effects of MOR pathway, we further found that PEBP overexpression caused a significant reduction of basal cAMP level and a significant inhibition of forskolin-induced cAMP elevation, and furthermore, PEBP overexpression significantly attenuated DAMGO-induced inhibition against forskolin-induced cAMP elevation and DAMGO-induced ERK activation. However, naloxone-precipitated withdrawal indicated by cAMP overshoot after sustained morphine treatment. These results indicated that PEBP could regulate AC through a mechanism independent of receptor, and PEBP overexpression inhibited DAMGO-induced ERK activation by aggregated inhibition on Raf-1. However, the upregulation of PEBP did not contribute to chronic morphine-induced compensation of AC activity. In summary, the following conclusions are drawn: (1) during the development of opioid dependence, addiction memory-related ERK regulation is not mediated by PEBP; (2) in cellular level, PEBP was not involved in MOR-mediated ERK regulation, which was possibly duo to little activation of PKC; (3) in cellular level, PEBP overexpression inhibited DAMGO-induced ERK activation and cAMP inhibiton, but did not affect other receptor properties, indicating that PEBP can regulate MOR functions in some aspects.Taken together, though PEBP has a potential role in regulating ERK and GPCR function, our findings in this paper suggest that PEBP does not contribute to those important elements in opioid dependence, including associated memory-induced ERK regulation and MOR activation-induced impotant biochemical events. PEBP overexpression inhibits intracellular basal cAMP and Raf-1, resulting in affection on DAMGO-induced cAMP inhibition and ERK activation, however, analysis in a general view, there is little chance that PEBP contributes to the mechanisms of opioid dependence through affecting opioid receptor-related signal transduction. Finally, further studies are needed to clarify the mechanisms of AC inhibition by PEBP independent of receptors.