KOR mRNA Research Articles

Prelimbic cortex dynorphin/κ opioid receptor system modulates methamphetamine-induced cognitive impairment.

Chronic exposure to methamphetamine (METH) causes severe and persistent cognitive impairment. The present study aimed to investigate the role of dynorphin/κ opioid receptor (KOR) system in the development of METH-induced cognitive impairment. We found that mice showed significant cognitive impairment in the novel object recognition test (NOR) following daily injections of METH (10mg/kg) for seven consecutive days. Systemic blockade of KOR prevented METH-induced cognitive impairment by pretreatment of the selective KOR antagonist norBNI (10mg/kg, i.p.) or KOR deletion. Then, significant increased dynorphin and KOR mRNA were observed exclusively in prelimbic cortex (PL) other than infralimbic cortex. Finally, microinjection with norBNI into PL also improved cognitive memory in METH-treated mice using NOR and spontaneous alternation behaviour test. Our results demonstrated that dynorphin/KOR system activation in PL may be a possible mechanism for METH-induced cognitive impairment and shed light on KOR antagonists as a potential neuroprotective agent against the cognitive deficits induced by drug abuse.

Effect of Analgesic Opioid Drugs on Opioid Receptor Genes Expression in HL-1 Mouse Cardiac Myocytes

BACKGROUND: The opioid system was mainly involved three types of opioid receptors (ORs): μ (MOR), δ (DOR) and κ (KOR). These ORs are activated by its agonist, a family of endogenous peptides: Endorphins, enkephalins, and dynorphins, respectively. AIM: This study determined the OR mRNA on effects of agonists exogenous morphine, fentanyl, D-penicillamine (2,5) enkephalin, and ketazocine in HL-1 mouse cardiac myocytes. MATERIALS AND METHODS: HL-1 mouse cardiac myocytes were treated with 10 μM morphine sulfate, 1 μM fentanyl,1 μM D-penicillamine (2,5) enkephalin, and 1 μM ketazocine. Total mRNAs were extracted and cDNA was synthesized and quantitative real-time polymerase chain reaction was used to analyze gene expression. RESULTS: The data analysis of MOR, DOR and KOR mRNA expression on effect of morphine was shown less level than control (0.61-fold, 0.67-fold, and 0.65-fold), respectively. The morphine-induced ORs down-regulation, whereas enkephalin treatment demonstrated highly significantly increased in mRNA of DOR (6.3-fold, p = 0.002). As well as, KOR mRNA expression was found highly significant increased under effect of Ketazocine (7.16-fold, p = 0.004). CONCLUSION: This study found DOR and KOR, but not MOR expressed in HL-1 mouse cardiac myocytes under activation of exogenous opioid analogists. These findings suggested that exogenous analogist’s opioids mimeses the endogenous analogist’s opioids.

κ-opioid receptor is involved in electroacupuncture analgesia via inhibition of spinal microglial Toll-like receptor 4 in neuropathic pain rats

To observe the effect of electroacupuncture (EA) on the expression of lumbar spinal κ-opioid receptor (KOR) and Toll-like receptor 4(TLR4) in microglia in neuropathic pain rats, so as to explore the role of cross-talk between KOR and TLK4 in EA-induced alleviation of chronic neuropathic pain. Wistar male rats were randomized into control, model, EA and EA plus KOR inhibitor (EA+inhibitor) groups (n=18 in each group). The neuropathic pain model was established in rats by ligature of the right sciatic nerve. EA was applied at bilateral "Zusanli"(ST36) and "Yanglingquan"(GB34) for 30 min, once daily for 5 days. JDTic dihydrochloride (a KOR inhibitor) was administrated by intraperitoneal injection before EA intervention. The difference value of paw withdrawal thermal latency (PWLD) of the bilateral hind-limbs was used as the thermal pain reaction level. At the end of experiments, the rat's lumbar spinal cord (L2-L4) was taken for detecting the expression of CD68 mRNA (a marker of the activated microglia) and Iba-1 (a marker for the activated and resting microglia) immunoactivity, and dynorphin content, and KOR mRNA and TLR4 protein (in immunomagnetic microbead method separated microglia) by using fluorescence quantitative PCR, immunofluorescence, radioimmunoassay and Western blot, separately. Compared with the control group, a strong thermal hyperalgesia was induced, the expression levels of Iba-1 and CD68 mRNA in the spinal cord, TLR4 protein of the spinal microglia were significantly increased(P<0.01) in the model group. The microglia were characterized by somatic hypertrophy and thickened branches in the model group. After EA intervention, the PWLD, the expression of Iba-1, CD68 mRNA and TLR4 protein of the microglia were significantly decreased(P<0.05), while the content of spinal dynorphin and the expression of KOR mRNA of the microglia increased in the EA group relative to the model group(P<0.05). The hypertrophic microglia shrinked slightly in the EA group. After injection of KOR inhibitor, the PWLD and expression levels of Iba-1, CD68 mRNA and TLR4 protein were significantly increased(P<0.05), and the expression of KOR mRNA was significantly decreased(P<0.05) in the EA+inhibitor group in comparison with the EA group. The analgesia effect of EA may partly mediated by spinal microglial KOR and the activation of KOR of microglia may be a target for inhibition of microglial TLR4-induced pro-inflammatory signaling.

Locomotor and anti-immobility effects of buprenorphine in combination with the opioid receptor modulator samidorphan in rats

Modulation of the opioid system has re-emerged as a potential therapeutic avenue for treating depression, with efficacy of a fixed-dose combination of buprenorphine (BUP), a partial μ-opioid receptor (MOR) agonist and κ-opioid receptor (KOR) antagonist, and samidorphan (SAM), a potent MOR antagonist, as an adjuvant treatment in patients with major depressive disorder (MDD). To advance understanding of the mechanism of action underlying this combination, we examined BUP, SAM and their combination in a series of rat behavioural assays. We examined effects on locomotor activity in Sprague Dawley (SD) rats over an extended period of time in a home-cage tracking system, and behavioural despair (immobility) in the forced swim test (FST), a commonly-used test to study antidepressants, in SD and Wistar-Kyoto (WKY) rats. Strain differences in opioid receptor and prepropeptide mRNA expression in the brain (prefrontal cortex, amygdala, hippocampus and striatum) were examined using qRT-PCR. BUP produced locomotor hyperactivity in SD rats from 2 to 6 h following administration, which was attenuated by SAM. In SD rats, a low, but not a high, dose of SAM in combination with BUP counteracted swim-stress induced immobility. This effect was not seen with BUP alone. In contrast, BUP alone reduced immobility in WKY rats, and this effect was enhanced by a low, but not high, dose of SAM. In WKY rats, MOR mRNA expression was higher in the hippocampus and lower in the striatum vs. SD rats. KOR mRNA expression was higher in the amygdala and nociceptin receptor (NOP) mRNA expression was lower in the hippocampus vs. SD rats. Differences in opioid receptor expression may account for the differential behavioural profile of WKY and SD rats. In summary, administration of BUP, a MOR receptor agonist together with a MOR opioid-receptor antagonist, SAM, reduces behavioural despair in animal models traditionally used to study effects of antidepressants.

Prenatal ethanol induces an anxiety phenotype and alters expression of dynorphin & nociceptin/orphanin FQ genes

Animal models have suggested that prenatal ethanol exposure (PEE) alters the κ opioid receptor system. The present study investigated the brain expression of dynorphin and nociceptin/orphanin FQ related genes and assessed anxiety-like behavior in the light-dark box (LDB), shelter-seeking and risk-taking behaviors in the concentric square field (CSF) test, and ethanol-induced locomotion in the open field (OF), in infant or adolescent Wistar rats that were exposed to PEE (0.0 or 2.0 g/kg, intragastrically, gestational days 17–20). We measured brain mRNA levels of prodynorphin (PDYN), κ opioid receptors (KOR), the nociceptin/orphanin FQ opioid peptide precursor prepronociceptin (ppN/OFQ) and nociceptine/orphanin FQ receptors (NOR). Prenatal ethanol exposure upregulated PDYN and KOR mRNA levels in the ventral tegmental area (VTA) in infant and adolescent rats and KOR mRNA levels in the prefrontal cortex in infant rats. The changes in gene expression in the VTA were accompanied by a reduction of DNA methylation at the PDYN gene promoter, and by a reduction of DNA methylation at the KOR gene promoter. The PEE-induced upregulation of PDYN/KOR in the VTA was accompanied by lower NOR gene expression in the VTA, and lower PDYN gene expression in the nucleus accumbens. PEE rats exhibited hypolocomotion in the OF, greater avoidance of the white and brightly lit areas in the LDB and CSF, and greater preference for the sheltered area in the CSF test. These results suggest that PEE upregulates the dynorphin system, resulting in an anxiety-prone phenotype and triggering compensatory responses in the nociceptin/orphanin FQ system. These findings may help elucidate the mechanisms that underlie the effects of PEE and suggest that the dynorphin and nociceptin/orphanin FQ systems may be possible targets for the prevention and treatment of PEE-induced alterations.

Heterodimerization of apelin and opioid receptors and cardiac inotropic and lusitropic effects of apelin in 2K1C hypertension: Role of pERK1/2 and PKC

AimsKappa Opioid receptors (KORs) change the impact of apelin on the phosphorylated ERK1/2 (pERK1/2). However, the role of interaction between KOR and apelin receptors (APJ) on the cardiac contractility effects of apelin and in regulation of pERK1/2 and PKC in the heart of renovascular hypertensive (2K1C) rats is unknown. Main methodsHemodynamic factors, the heterodimerization of KOR and APJ, the expression of KOR mRNA and protein and pERK1/2 in the left ventricle of 2K1C rats were measured following APJ, KOR, PKC and Gi path inhibition by F13A, nor-BNI, chelerythrine and PTX respectively. Key findingsApelin in 40 and 60μg/kg doses increased cardiac contractility, and reduced mean arterial pressure. The cardiac impacts in both doses were reduced by F13A, nor-BNI and chelerytrine and blocked by PTX. Hypertension increased the expression of KORs and heterodimerization of APJ and KOR, and reduced pERK1/2 in the left ventricle. Apelin, in both doses reduced (normalized) heterodimerization and recovered the reduction in pERK1/2. The recovery of ERK1/2 phosphorylation was accompanied by reduction of KOR and APJ heterodimerization. Significance2K1C hypertension increased the expression of KORs and heterodimerization of APJ and KORs. The heterodimerization was associated by reduction of ERK phosphorylation and altered the cardiac inotropic and lusitropic effects of apelin. These changes may participate in pathophysiology of cardiac dysfunction in renovascular hypertension that is associated with subnormal level of serum apelin. Apelin- induced recovery of ERK1/2 phosphorylation and KOR-APJ dimerization may nominate apelin as a therapeutic goal in treatment of this kind of hypertension.

Down-regulation of the tumour suppressor \u03ba-opioid receptor predicts poor prognosis in hepatocellular carcinoma patients

BackgroundOpioid receptors have become increasingly implicated in cancer progression and long-term patient outcomes. However, the expression and significance of the κ-opioid receptor (KOR) in hepatocellular carcinoma (HCC) remain unclear.MethodsIn this study, KOR mRNA expression was analysed by real-time quantitative PCR in 64 pairs of HCC tumour tissues and adjacent non-tumour tissues, and KOR protein expression was analysed by immunohistochemistry in 174 HCC patients. We investigated the correlation between KOR expression and clinicopathological parameters to illustrate the potential prognostic significance of KOR expression in HCC.ResultsKOR mRNA expression was significantly down-regulated in 79.69% (51 of 64) of the HCC tumour samples, and KOR expression in tumour tissue was significantly lower than that in adjacent non-tumour tissues (P < 0.001). ROC curve analysis showed that KOR mRNA expression yielded AUC of 0.745, for the detection of HCC patients. Low KOR mRNA expression in HCC was correlated with aggressive clinicopathological parameters, such as tumour size (P = 0.015), differentiation grade (P = 0.011), and TNM stage (P = 0.021). Moreover, down-regulation of KOR protein expression in HCC tissues was detected in 174 HCC patients. Similarly, negative KOR protein expression was significantly correlated with aggressive clinicopathological features, such as tumour size (P = 0.002), vascular invasion (P = 0.003), differentiation grade (P = 0.026), and TNM stage (P = 0.030). Furthermore, Kaplan-Meier survival analysis demonstrated that down-regulation of KOR in HCC indicated poor prognosis. KOR deficiency (KORT < N) was correlated to a shorter survival rate and an increased recurrence (both P < 0.001). In univariate and multivariate survival analyses, KOR was identified as a promising independent risk factor for both overall survival (OS, both P < 0.001) and recurrence-free survival (RFS, both P < 0.001).ConclusionsDown-regulation of KOR in HCC tumour tissues has a strong association with poor prognosis and KOR might be a potential tumour suppressor.

Chronic nicotine-induced changes in gene expression of delta and kappa-opioid receptors and their endogenous ligands in the mesocorticolimbic system of the rat.

Delta and kappa opioid receptors (DOR and KOR, respectively) and their endogenous ligands, proenkephalin (PENK) and prodynorphin (PDYN)-derived opioid peptides are proposed as important mediators of nicotine reward. This study investigated the regulatory effect of chronic nicotine treatment on the gene expression of DOR, KOR, PENK and PDYN in the mesocorticolimbic system. Three groups of rats were injected subcutaneously with nicotine at doses of 0.2, 0.4, or 0.6 mg/kg/day for 6 days. Rats were decapitated 1 hr after the last dose on day six, as this timing coincides with increased dopamine release in the mesocorticolimbic system. mRNA levels in the ventral tegmental area (VTA), lateral hypothalamic area (LHA), amygdala (AMG), dorsal striatum (DST), nucleus accumbens, and medial prefrontal cortex were measured by quantitative real-time PCR. Our results showed that nicotine upregulated DOR mRNA in the VTA at all of the doses employed, in the AMG at the 0.4 and 0.6 mg/kg doses, and in the DST at the 0.4 mg/kg dose. Conversely, PDYN mRNA was reduced in the LHA with 0.6 mg/kg nicotine and in the AMG with 0.4 mg/kg nicotine. KOR mRNA was also decreased in the DST with 0.6 mg/kg nicotine. Nicotine did not regulate PENK mRNA in any brain region studied.

Molecular identification of the Dyn/Kor system and its potential role in the reproductive axis of goldfish

To ascertain the significance of the dynorphin/kappa-opioid receptor (Dyn/Kor) system in fish reproduction, prodynorphin (pdyn) cDNA was cloned from goldfish. Two Dyn peptides (DynA and DynB) are present in the goldfish prodynorphin precursor. Both DynA and DynB are biologically active as they are able to functionally interact with the goldfish Kor expressed in cultured eukaryotic cells to suppress forskolin-induced CRE promoter activity. RT-PCR analysis showed that pdyn is widely expressed in brain regions, with the highest expression in hypothalamus. During ovarian development, hypothalamic pdyn and kor mRNA levels are lower in the early vitellogenic stage. Then the biological effects of Dyn peptides on salmon gonadotropin releasing hormone (sgnrh), luteinizing hormone beta (lhb) and follicle stimulating hormone beta (fshb) mRNA synthesis were further investigated in goldfish. Intraperitoneal injections of DynA and DynB significantly reduced hypothalamic sgnrh and pituitary lhb and fshb mRNA levels in male goldfish, but these two peptides only down-regulated sgnrh and lhb mRNA expression in female goldfish. In vitro studies revealed that DynA also decreased lhb mRNA levels in primary cultures of pituitary cells, indicating that this peptide can exert its actions at the pituitary level. Our findings suggest that the Dyn/Kor system plays a negative role in regulating the reproductive axis in goldfish.

Electroacupuncture relieves labour pain and influences the spinal dynorphin/κ-opioid receptor system in rats.

The dynorphin (DYN)/κ-opioid receptor (KOR) system plays a key role in the control of labour pain. Our previous clinical study reported that electroacupuncture (EA) provided intrapartum analgesia, but the underlying mechanisms of action have not been fully elucidated. To observe the effect of EA on labour pain and to explore the underlying mechanisms of action in a rat model. Copulation-confirmed pregnant rats (n=120) were given castor oil to induce labour. Rats remained untreated (control group, n=20) or received either meperidine (an opioid that is commonly used to treat labour pain, n=20) or EA at SP6, LI4, SP6+LI4 or SP10 (four groups, n=20 each). Labour pain was evaluated by the warm water tail-flick test. Serum DYN values were measured by ELISA. Protein and mRNA expression of prodynorphin (PDYN, the precursor protein of DYN) and KOR were analysed by Western blotting and real-time PCR, respectively. EA treatment at all acupuncture point combinations studied significantly relieved labour pain and increased serum DYN concentrations, to a degree similar to that achieved with meperidine. EA notably enhanced protein expression of KOR and PDYN and mRNA expression in the lumbar spinal cord but not in the cerebral cortex. The size of effect varied by EA group in the order: SP6>LI4>SP6+LI4>SP10 for all parameters measured, indicating differential effects relating to acupuncture point selection/combination. The present study indicates that EA relieves labour pain, at least in part, by regulation of the spinal DYN/KOR system in a rat model.

κ-Opioid Receptor Is Colocalized in GnRH and KNDy Cells in the Female Ovine and Rat Brain.

Kisspeptin-neurokinin B-dynorphin (KNDy) cells of the hypothalamus are a key component in the neuroendocrine regulation of GnRH secretion. Evidence in sheep and other species suggests that dynorphin released by KNDy cells inhibits pulsatile GnRH secretion by acting upon κ-opioid receptors (KOR). However, the precise anatomical location and neurochemical phenotype of KOR-expressing cells in sheep remain unknown. To this end, we determined the distribution of KOR mRNA and protein in the brains of luteal phase ewes, using an ovine specific KOR mRNA probe for in situ hybridization and an antibody whose specificity we confirmed by Western blot analyses and blocking peptide controls. KOR cells were observed in a number of regions, including the preoptic area (POA); anterior hypothalamic area; supraoptic and paraventricular nuclei; ventromedial, dorsomedial, and lateral hypothalamus; and arcuate nucleus. Next, we determined whether KOR is colocalized in KNDy and/or GnRH cells. Dual-label immunofluorescence and confocal analysis of the KNDy population showed a high degree of colocalization, with greater than 90% of these neurons containing KOR. Surprisingly, GnRH cells also showed high levels of colocalization in sheep, ranging from 74.4% to 95.4% for GnRH cells in the POA and medial basal hypothalamus, respectively. Similarly, 97.4% of GnRH neurons in the POA of ovariectomized, steroid-primed female rats also contained immunoreactive KOR protein. These findings suggest that the inhibitory effects of dynorphin on pulsatile GnRH secretion may occur either indirectly by actions upon KOR within the KNDy population and/or directly via the activation of KOR on GnRH cells.

Stress-induced epigenetic regulation of κ-opioid receptor gene involves transcription factor c-Myc

Exposure to stress is associated with adverse emotional and behavioral responses. Whereas the κ-opioid receptor (KOR) system is known to mediate some of the effects, it is unclear whether and how stress affects epigenetic regulation of this gene. Because the KOR gene can use two promoters (Pr1 and Pr2) and two polyadenylation signals (PA1 and PA2), it is also interesting whether and how these distinct regulatory mechanisms are differentially modulated by stress. The current study examined the effects of stress on these different regulatory mechanisms of the KOR gene. Results showed that stress selectively increased the expression of KOR mRNA isoforms controlled by Pr1 and terminated at PA1 in specific brain areas including the medial-prefrontal cortex, hippocampus, brainstem, and sensorimotor cortex, but not in the amygdala or hypothalamus. These effects correlated with altered epigenetic state of KOR Pr1 chromatin, as well as elevation and increased recruitment of the principal transcription factor c-Myc, which could activate Pr1. Stress-induced modulation of Pr1 was further validated using glutamate-sensitive murine hippocampal cell line, HT22. The results revealed a common molecular mechanism underlying the effect of stress on selected chromatin regions of this gene at the cellular level and in the context of whole animal and identified a critical role for c-Myc in stress-triggered epigenetic regulation of the KOR gene locus. This study sheds light on the mechanisms of stress-induced epigenetic regulation that targets specific chromatin segments and suggests certain KOR transcripts and its principal transcription factor c-Myc as potential targets for brain-area-specific intervention.

Opioid receptor expression in human brain and peripheral tissues using absolute quantitative real-time RT-PCR

BackgroundThe actions of endogenous opioid peptides are mediated by 3 main classes of opioid receptors; mu (MOR), kappa (KOR), and delta (DOR). MethodsWe developed an absolute quantitative real-time reverse transcriptase PCR (AQ-rt-RT-PCR) assay to quantify MOR, DOR, and KOR mRNA in 22 human tissues. ResultsMOR mRNA was greatly enriched (12–20×106copies/μg) in the cerebellum, nucleus accumbens, and caudate nucleus; moderate (6×106copies/μg) in the dorsal root ganglion, spinal cord, and adrenal gland; low (2×104copies/μg) in the pancreas and small intestine; and absent in the lung, spleen, kidney, heart, skeletal muscle, liver, and thymus. High levels (>8.8×106copies/μg) of DOR mRNA were expressed in the brain and dorsal root ganglion; moderate (1.5×106copies/μg) in the adrenal gland and pancreas; low (2×104–6.5×105copies/μg in the cerebellum, spinal cord, small intestine, skeletal muscle, thymus, lung, and kidney); and very low (3.8×103copies/μg) in the heart. DOR mRNA was not detected in the spleen or liver. KOR mRNA was moderate (1×106copies/μg) in brain regions and dorsal root ganglion, but low (1.6–7×105copies/μg) in the cerebellum, temporal lobe and all other peripheral tissues. ConclusionsOur data demonstrate that the AQ-rt-RT-PCR is a highly reproducible and precise method to study the expression of opioid receptors in various tissues and under different disease conditions.

Dual action of epidermal growth factor: extracellular signal-stimulated nuclear–cytoplasmic export and coordinated translation of selected messenger RNA

We report the first example of a coordinated dual action of epidermal growth factor (EGF) in stimulating the nuclear-cytoplasmic export and translation of a select messenger RNA (mRNA). The effect of EGF is mediated by the RNA-binding protein Grb7 (growth factor receptor-bound protein 7), which serves as an adaptor for a specific mRNA-protein export complex and a translational regulator. Using the kappa-opioid receptor (OR [KOR]) as a model, we demonstrate that EGF activates nuclear SHP-2 (Src homology region 2-containing tyrosine phosphatase), which dephosphorylates Grb7 in the nucleus. Hypophosphorylated Grb7 binds to the KOR mRNA and recruits the Hu antigen R-exportin-1 (CRM1) complex to form a nuclear-cytoplasmic export complex that exports KOR mRNA. EGF also activates focal adhesion kinase in the cytoplasm to rephosphorylate Grb7, releasing KOR mRNA for active translation. In summary, this study uncovers a coordinated, dual activity of EGF in facilitating nuclear export of a specific mRNA-protein complex as well as translational activation of the exported mRNA.

EGF takes dual control of mRNA

EGF coordinates the nuclear and cytoplasmic activities of an RNA-binding protein to ensure a specific mRNA is translated at the right time and place in neurons, say [Tsai et al.][1] ![Figure][2] KOR mRNA (green) is largely confined to the nucleus (blue) in neurons lacking the EGF-regulated

Copb1-facilitated axonal transport and translation of kappa opioid-receptor mRNA.

mRNA of kappa opioid receptor (KOR) can be transported to nerve fibers, including axons of dorsal root ganglia (DRG), and can be locally translated. Yeast three-hybrid screening identifies Copb1 as a kor mRNA-associated protein that form complexes with endogenous kor mRNA, which are colocalized in the soma and axons of DRG neurons. Axonal transport of kor mRNA is demonstrated, directly, by observing mobilization of biotin-labeled kor mRNA in Campenot chambers. Efficient transport of kor mRNA into the side chamber requires Copb1 and can be blocked by a drug that disrupts microtubules. The requirement for Copb1 in mobilizing kor mRNA is confirmed by using the MS2-GFP mRNA-tagging system. Furthermore, Copb1 also facilitates the translation of kor mRNA in the soma and axons. This study provides evidence for a microtubule-dependent, active axonal kor mRNA-transport process that involves Copb1 and can stimulate localized translation and suggests coupling of transport and translation of mRNAs destined to the remote areas such as axons.

The adaptor Grb7 links netrin-1 signaling to regulation of mRNA translation

We previously reported a novel biological activity of Netrin-1 in translational stimulation of kappa opioid receptor (KOR). We now identify Grb7 as a new RNA-binding protein that serves as the molecular adaptor for transmitting Netrin-1 signals, through focal adhesion kinase (FAK), to the translation machinery. Grb7 binds specifically to the first stem loop of kor mRNA 5'-UTR through a new RNA-binding domain located in its amino terminus. Upon binding to its capped, target mRNA, Grb7 blocks the recruitment of eIF4E, rendering mRNA untranslatable. The RNA-binding and translation-repressive activity is reduced by FAK-mediated hyperphosphorylation on two tyrosine residues of its carboxyl terminus. This study reports an adaptor protein Grb7 that transmits the stimulating signals of Netrin-1 to the translational machinery to rapidly regulate mRNA translation.

Axonal mRNA transport and localized translational regulation of kappa-opioid receptor in primary neurons of dorsal root ganglia.

kappa-opioid receptor (KOR) is detected pre- and postsynaptically, but the subcellular localization, translation, and regulation of kor mRNA in presynaptic compartments of sensory neurons remain elusive. In situ hybridization detected axonal distribution of kor mRNA in primary neurons of dorsal root ganglia (DRG). The MS2-fused GFP tracked kor mRNA transport from DRG neuronal soma to axons, requiring its 5' and 3' UTRs. In Campenot chambers, axonal translation of kor mRNA was demonstrated for DRG neurons, which depended on its 5' UTR and was stimulated by KCl depolarization. KCl depolarization of DRG neurons rendered redistribution of kor mRNA from the postpolysomal fraction to the translationally active polysomal fraction. This study provided evidence for mRNA transport and regulation of presynaptic protein synthesis of nonstructural proteins like KOR in primary sensory neurons and demonstrated a mechanism of KCl depolarization-stimulated axonal mRNA redistribution for localized translational regulation.

Netrin-1 Signaling RegulatesDe NovoProtein Synthesis of κ Opioid Receptor by Facilitating Polysomal Partition of Its mRNA

The expression of kappa opioid receptor (KOR) is subjected to both transcriptional and posttranscriptional controls. We report that KOR translation is regulated by netrin-1 in primary neurons of dorsal root ganglion (DRG) and in P19 embryonal carcinoma cells. Without stimulation, a significant portion of KOR mRNA is maintained in a dormant state and partitions in the translationally inactive, post-polysomal fraction. During netrin-1 stimulation, which activates its downstream target focal adhesion kinase (FAK), KOR mRNA rapidly partitions to the translationally active polysomal fraction. Functionally, the newly synthesized KOR proteins in DRG neurons are able to bind to specific ligands. This report describes the first example of netrin-1 signaling in the translational control of a drug receptor KOR, which involves the mediator of netrin-1, FAK, and a novel mechanism that enhances the association of target mRNA with polysomes for translational activation.

Morphine-induced changes in the activity of proopiomelanocortin and prodynorphin systems in zymosan-induced peritonitis in mice

We have shown that supplementation of proinflammatory agent with a high dose of morphine not only abolishes inflammation-related pain symptoms but also inhibits influx of leukocytes to the inflamed peritoneal cavity. Present investigations focused on effects of morphine on proopiomelanocortin and prodynorphin systems during zymosan-induced peritonitis. Males of SWISS mice were ip injected with zymosan (Z, 40 mg/kg) or zymosan with morphine (ZM, 20 mg/kg). At time 0 (controls) and 4 and 24 h after stimulation, peritoneal leukocytes (PTLs) were counted, PTL levels of opioid peptides (β-endorphin and dynorphin) measured by radioimmunoassays, while mRNAs coding their respective precursors (POMC and PDYN) and receptors (MOR and KOR) determined by QRT-PCR. Influx of inflammatory PTLs, mainly PMNs, was significantly delayed by morphine co-injection. Total levels of β-endorphin and dynorphin corresponded with PTL numbers, while levels per cell were similar in all groups except of β-endorphin, decreased in ZM at 4 h. Levels of both peptides in peritoneal fluid were increased in Z and ZM groups at 4 h, while at 24 h only in case of β-endorphin in Z group. POMC was increased only in ZM group at 4 h of peritonitis, while PDYN in both Z and ZM groups at the same time. MOR mRNA was increased 24 h after injection in Z and ZM groups, while KOR mRNA was similar in all groups except of decrease in Z at 24 h. In conclusion, endogenous opioids and their receptors are involved in zymosan-induced peritonitis and affected in various ways by morphine co-injection.