D-Ala2,D-Leu5]enkephalin Research Articles

Contribution of organic anion transporting polypeptide OATP-C to hepatic elimination of the opioid pentapeptide analogue [D-Ala2, D-Leu5]-enkephalin.

The objective of this study was to examine the transport activity of the human organic anion transporter OATP-C (SLC21A6) for oligopeptides that are eliminated rapidly from the systemic circulation. We focused on an opioid peptide analogue, [D-Ala(2), D-Leu(5)]-enkephalin (DADLE), a linear pentapeptide modified to be stable. [(3)H]DADLE was taken up by rat isolated hepatocytes in a saturable manner and highly accumulated in the liver after intravenous administration to rats. The uptake of [(3)H]DADLE by the isolated hepatocytes was inhibited by several organic anions and pentapeptides, but not by tetra- or tripeptides. When OATP-C was expressed in Xenopus laevis oocytes, a significant increase in uptake of [(3)H]DADLE was observed. Moreover, the inhibitory effects of various compounds, including some peptides, on [(3)H]estrone-3-sulfate uptake by OATP-C were similar to those observed in [(3)H]DADLE uptake by rat isolated hepatocytes. In conclusion, it was demonstrated that OATP-C contributes to the rapid hepatic excretion of peptides and peptide-mimetic drugs.

Effects of intracerebroventricular application of the delta opioid receptor agonist [d-Ala2, d-Leu5] enkephalin on neurological recovery following asphyxial cardiac arrest in rats

The delta opioid receptor (DOR) agonist [d-Ala2, d-Leu5] enkephalin (DADLE) has been implicated as a novel neuroprotective agent in the CNS. The current study was designed to evaluate the effects of intracerebroventricular (ICV) application of DADLE on neurological outcomes following asphyxial cardiac arrest (CA) in rats. Male Sprague-Dawley rats were randomly assigned to four groups: Sham group, CA group, DADLE group (DADLE+CA), and Naltrindole group (Naltrindole and DADLE+CA). All drugs were administered into the left cerebroventricle 30 min before CA. CA was induced by 8-min asphyxiation and the animals were resuscitated with a standardized method. DOR protein expression in the hippocampus was significantly increased in the CA group at 1 h after restoration of spontaneous circulation (ROSC) compared with the Sham group. As time progressed, expression of DOR proteins decreased gradually in the CA group. Treatment with DADLE alone or co-administration with Naltrindole reversed the down-regulation of DOR proteins in the hippocampus induced by CA at 24 h after ROSC. Compared with the CA group, the DADLE group had persistently better neurological functional recovery, as assessed by neurological deficit score (NDS) and Morris water maze trials. The number of surviving hippocampal CA1 neurons in the DADLE group was significantly higher than those in the CA group. However, administration of Naltrindole abolished most of the neuroprotective effects of DADLE. We conclude that ICV administration of DADLE 30 min before asphyxial CA has significant protective effects in attenuating hippocampal CA1 neuronal damage and neurological impairments, and that DADLE executes its effects mainly through DOR.

Delta opioid agonist [D-Ala2, D-Leu5] enkephalin (DADLE) reduced oxygen–glucose deprivation caused neuronal injury through the MAPK pathway

It has been demonstrated that [D-Ala2, D-Leu5] enkephalin (DADLE), a delta opioid agonist, protected neuron from hypoxic neuronal injury by activating the delta opioid receptor (DOR). However, whether DADLE can prevent neuronal injury induced by severe hypoxia like oxygen–glucose deprivation (OGD) is not clear. Here, we investigated whether DADLE has a protective effect against neuronal injury induced by oxygen–glucose deprivation. Neuron viability was measured by MTT and neuron injury was assessed by lactate dehydrogenase (LDH) release. Protein expression was examined by Western blot. The results showed that DADLE protected the cortical neuron in a dose-dependent way from OGD injury. And this neuroprotective effect could be completely blocked by delta2 opioid antagonist Naltrindole. DADLE increased phosphorylation of ERK and prevented OGD-induced p38 phosphorylation. Neither DADLE nor Naltrindole had any appreciable effect on phosphorylation of JNK. One of the protective mechanisms of DADLE on OGD neurons may be due to the dynamic balance between the activation of ERK and the p38.

Hibernation-like state induced by an opioid peptide protects against experimental stroke

BackgroundDelta opioid peptide [D-ala2,D-leU5]enkephalin (DADLE) induces hibernation in summer ground squirrels, and enhances preservation and survival of isolated or transplanted lungs and hearts. In the present study, we investigated the protective effect of DADLE in the central nervous system.ResultsAdult Sprague-Dawley rats were pretreated with DADLE (4 mg/kg every 2 h × 4 injections, i.p.) or saline prior to unilateral occlusion of the middle cerebral artery (MCA). Daily behavioral tests revealed that ischemic animals treated with DADLE did not show any significant behavioral dysfunctions compared with saline-treated ischemic animals. Opioid antagonists only transiently inhibited the protective effect of DADLE, indicating the participation of non-opioid mechanisms in DADLE neuroprotection. Histological examination using triphenyltetrazolium chloride (TTC) revealed that brains from ischemic animals treated with DADLE, either alone or with adjuvant opioid blockers, exhibited almost completely intact striata. In contrast, brains from ischemic animals that received saline showed significant infarction in the lateral striatum. Analyses of apoptotic cell death revealed a significant increase in the p-53 mRNA expression in the striatum of ischemic animals that received saline, while those that received DADLE exhibited near normal striatal p-53 expression. This protective effect was accompanied by significant increments in protein levels of glial cell line-derived neurotrophic factor in the striatum of DADLE-treated ischemic animals.ConclusionThese results indicate that DADLE protected against necrotic and apoptotic cell death processes associated with ischemia-reperfusion injury. The present study demonstrates that delta opioids are crucially involved in stroke, suggesting that the opioid system is important in the study of brain injury and protection.

δ-Opioid receptors activate ERK/MAP kinase via integrin-stimulated receptor tyrosine kinases

Integrin-mediated cell adherence to extracellular matrix proteins results in stimulation of ERK1/2 activity, a mechanism involving focal adhesion tyrosine kinases (pp125FAK, Pyk-2) and epidermal growth factor receptors (EGFRs). G protein-coupled receptors (GPCRs) may also mediate ERK1/2 activation in an integrin-dependent manner, the underlying signaling mechanism of which still remains unclear. Here we demonstrate that the δ-opioid receptor (DOR), a typical GPCR, stimulates ERK1/2 activity in HEK293 cells via integrin-mediated transactivation of EGFR function. Inhibition of integrin signaling by RGDT peptides, cytochalasin, and by keeping the cells in suspension culture both blocked [D-Ala2, D-Leu5]enkephalin (DADLE)- and etorphine-stimulated ERK1/2 activity. Integrin-dependent ERK1/2 activation does not involve FAK/Pyk-2, because over-expression of the FAK/Pyk-2 inhibitor SOCS-3 failed to attenuate DOR signaling. Exposure of the cells to the EGFR inhibitors AG1478 and BPIQ-I blocked DOR-mediated ERK1/2 activation. Because RGDT peptides also prevented DOR-mediated EGFR activation, the present findings indicate that in HEK293 cells DOR-stimulated ERK1/2 activity is mediated by integrin-stimulated EGFRs. Further studies with the phospholipase C (PLC) inhibitors U73122 and ET-18-OCH3 revealed that opioid-stimulated integrin activation is sensitive to PLC. In contrast, integrin-mediated transactivation of EGFR function appears to be dependent on PKC-δ, as indicated by studies with rottlerin and siRNA knock-down. A similar ERK1/2 signaling pathway was observed for NG108-15 cells, a neuronal cell line endogenously expressing the DOR. In these cells, the nerve growth factor TrkA receptor replaces the EGFR in connecting DOR-activated integrins to the Ras/Raf/ERK1/2 pathway. Together, these data describe an alternative ERK1/2 signaling pathway in which the DOR transactivates the growth factor receptor associated mitogen-activated protein kinase cascade in an integrin-dependent manner.

δ-Opioid receptors stimulate ERK1/2 activity in NG108-15 hybrid cells by integrin-mediated transactivation of TrkA receptors

This study demonstrates that activation of δ-opioid receptors (DORs) in neuroblastoma×glioma (NG108-15) hybrid cells by [d-Ala2, d-Leu5]enkephalin (DADLE) and etorphine significantly enhances cell adhesion to fibronectin-coated wells. This effect is blocked by both naloxone and integrin binding RGDT peptides. In addition, cell adhesion turned out to be a prerequisite for DOR-stimulated transactivation of Tropomyosin-related kinase A (TrkA) and extracellular signal-regulated kinases 1/2 (ERK1/2). Because inhibition of TrkA activation by AG879 completely blocked DOR- and integrin-mediated ERK1/2 signaling, the present results indicate that in NG108-15 cells DOR-stimulated ERK1/2 activation is mediated by integrin-induced transactivation of TrkA.

δ‐OPIOID RECEPTOR STIMULATION ENHANCES THE GROWTH OF NEONATAL RAT VENTRICULAR MYOCYTES VIA THE EXTRACELLULAR SIGNAL‐REGULATED KINASE PATHWAY

1. The aims of the present study were to determine whether delta-opioid receptor stimulation enhanced proliferation of and to investigate the role of the extracellular signal-regulated kinase (ERK) pathway in ventricular myocytes from neonatal rats. 2. At concentratins ranging from 10 nmol/L to 10 micromol/L, [D-Ala2,D-Leu5]enkephalin (DADLE) concentration-dependently promoted myocardial growth and DNA synthesis and altered the cytoskeleton. 3. At 1 micromol/L, DADLE also increased the expression and phosphorylation of ERK. 4. These effects of 1 micromol/L DADLE were abolished by 10 micromol/L naltrindole, a selective delta-opioid receptor antagonist, 10 nmol/L U0126, a selective ERK antagonist, 1 micromol/L staurosporine, an inhibitor of protein kinase (PK) C, and 100 micromol/L Rp-adenosine 3',5'-cyclic monophosphorothioate triethylammonium salt hydrate (Rp-cAMPS), an inhibitor of PKA. 5. In conclusion, delta-opioid receptor stimulation enhances the proliferation and development of the ventricular myocytes of neonatal rats. The ERK pathway and related signalling mechanisms, namely PKC and PKA, are involved.

Dose-dependent neuroprotection of delta opioid peptide [d-Ala2, d-Leu5] enkephalin in neuronal death and retarded behavior induced by forebrain ischemia in rats

Cerebral ischemic insult, mainly induced by cardiovascular disease, is one of the most severe neurological diseases in clinical. There's mounting evidence showing that delta opioid agonist [D-Ala2, D-Leu5] enkephalin (DADLE) has a tissue-protective effect. However, whether this property is effective to prevent neuronal death induced by forebrain ischemia is not clear. This study was aimed to investigate whether intracerebroventricular (ICV) administration of DADLE has a neuroprotective effect against forebrain ischemia in rats. We found in our study that administration of DADLE 45 min before forebrain ischemia had significant protective effect against CA1 neuronal lose. Further more, we found that DADLE had a dose-dependent protection for improving behavioral retardation revealed by Morris water maze and motor score test, while naltrindole, the antagonist of delta opioid receptor, partially abolished neuroprotective effect of DADLE, which implicated that both opioid and non-opioid systems are involved in ischemic insults and neuroprotection.

Effects of bilateral flocculus shutdown on the primate long-term adaptation of horizontal vestibulo-ocular reflex

The effects of nociceptin (orphanin FQ) on medial vestibular nucleus (MVN) neurons in vitro, and on vestibulo-ocular reflex (VOR) function in vivo, were investigated in order to determine the role of `opioid-like orphan' (ORL1) receptors in modulating vestibular reflex function in the rat. Nociceptin (100 nM–1 μM) potently inhibited the spontaneous discharge of the majority (86%) of MVN neurons tested in the rat dorsal brainstem slice preparation in vitro. This inhibition was dose-dependent and persisted after blockade of synaptic transmission in low Ca2+/Co2+ medium. The inhibitory effects were insensitive to the opioid antagonist naloxone, but were effectively antagonised by the selective ORL1 receptor antagonist, [Phe1Ψ(CH2–NH)Gly2]Nociceptin(1–13)NH2. The majority of MVN neurons (∼70%) were inhibited by both nociceptin and the δ-opioid receptor agonist, [d-ala2, d-leu5]-enkephalin (DADLE), while a minority of cells (∼30%) were selectively responsive either to DADLE or to nociceptin, but not both. Co-application of nociceptin and DADLE to neurons that were responsive to both agonists, resulted in an inhibitory response that was the same as or less than the inhibition evoked by either agonist alone. Intracellular whole-cell patch clamp recordings from identified Type A and Type B MVN cells showed that both these cell types are responsive to nociceptin, which induced membrane hyperpolarisation and decrease in input resistance consistent with its known effects on membrane K currents in other cell types. In alert rats, i.c.v. injection of nociceptin caused a significant decrease in the gain of the hVOR and resulted in a prolongation of post-rotatory nystagmus in darkness. The decrease in VOR gain and the increase in the VOR time-constant was significant even at low doses of nociceptin which did not cause other observable behavioural effects. These findings demonstrate that endogenously released nociceptin may have a hitherto unexplored role in the functional modulation of the neural pathways that mediate vestibular reflexes in vivo.

Delta opioid receptors stimulation with [ d-Ala 2, d-Leu 5] enkephalin does not provide neuroprotection in the hippocampus in rats subjected to forebrain ischemia

It has been reported that delta opioid agonists can have neuroprotective efficacy in the central nervous system. This study was conducted to test the hypothesis that a delta opioid receptor (DOR) agonist, [ d-Ala 2, d-Leu 5] enkephalin (DADLE), can improve neuron survival against experimental forebrain ischemia in rats. Using male rats ( n = 125), intraperitoneal injection of DADLE (0, 0.25, 1, 4, 16 mg kg −1) was performed 30 min before ischemia. Ten minutes interval forebrain ischemia was provided by the bilateral carotid occlusion combined with hypotension (35 mmHg) under isoflurane (1.5%) anesthesia. All animals were neurologically and histologically evaluated after a recovery period of 1 week. As histological evaluation, percentages of ischemic neurons in the CA1, CA3, dentate gyrus (DG) were measured. During the recovery period, 27 rats died because of apparent upper airway obstruction, seizure, or unidentified causes. There were no differences in the motor activity score among the groups. Ten minutes forebrain ischemia induced approximately 75, 20, and 10% neuronal death in the CA1, CA3, and DG, respectively. Any doses of DADLE did not attenuate neuronal injury in the hippocampus after ischemia. Pre-ischemic treatment of DORs agonism with DADLE did not provide any neuroprotection to the hippocampus in rats subjected to forebrain ischemia.

Picomolar concentrations of hibernation induction delta opioid peptide [D‐Ala2,D‐Leu5]enkephalin increase the nerve growth factor in NG‐108 cells

The delta opioid peptide [D-Ala2,D-Leu5]enkephalin (DADLE) has been shown to be a neuroprotective agent via mechanisms that are not totally understood. We previously demonstrated that the i.p. injection of DADLE in mice causes an increase of nerve growth factor (NGF) in the brain. To further clarify the NGF-increasing action of DADLE, we examined here the NGF-increasing effect of DADLE in vitro, using cultured NG-108 cells. DADLE dose-dependently increases the immunoreactive level of NGF in NG-108 cells in a bell-shape manner, with the effective DADLE concentrations in the picomolar range (0.01-100 pM). Also, DADLE at 1 pM selectively increases c-Jun and c-Fos, but not c-Rel. These results indicate that DADLE is one of the most potent agents in increasing the NGF in the biological system and that this action of DADLE involves selective increases of c-Jun and c-Fos, transcription factors that promote the NGF expression.

Delta-opioid Agonist SNC80 Can Attenuate the Development of Dynorphin A-induced Tactile Allodynia in Rats

Delta-opioid Agonist SNC80 Can Attenuate the Development of Dynorphin A-induced Tactile Allodynia in Rats

Ultrastructural and immunocytochemical analyses of opioid treatment effects on PC3 prostatic cancer cells.

Some opioid peptides are able to inhibit the growth of human prostatic cancer cells; in particular, the [D-Ala(2),D-Leu(5)] enkephalin (DADLE) reduces PC3 cell growth. In order to understand how DADLE decreases cell proliferation, we investigated, by electron microscopy, its effects on PC3 cellular components. PC3 cells were incubated with DADLE and processed for both ultrastructural morphology and immunoelectron microscopy. Some cells were incubated with BrU to determine the transcriptional rate. BrU and DADLE molecules were detected by immunogold techniques and the labeling was quantitatively evaluated. Modifications of some cytoplasmic and nuclear components were observed in DADLE-treated cells. Moreover, treated cells incorporated lower amounts of BrU than control cells. DADLE molecules were located in the cytoplasm and in the nucleus, especially on mRNA transcription and early splicing sites. Our data suggest that DADLE is able to slow down the synthetic activity of PC3 cells, perhaps interfering with nuclear functions.

Delta opioid peptide (D-Ala 2, D-Leu 5) enkephalin: linking hibernation and neuroprotection.

Hibernation is a potential protective strategy for the peripheral, as well as for the central nervous system. A protein factor termed hibernation induction trigger (HIT) was found to induce hibernation in summer-active ground squirrels. Purification of HIT yielded an 88-kD peptide that is enriched in winter hibernators. Partial sequence of the 88-kD protein indicates that it may be related to the inhibitor of metalloproteinase. Using opioid receptor antagonists to elucidate the mechanisms of HIT, it was found that HIT targeted the delta opioid receptors. Indeed, delta opioid (D-Ala 2, D-Leu 5) enkephalin (DADLE) was shown to induce hibernation. Specifically, HIT and DADLE were found to prolong survival of peripheral organs, such as the lung, the heart, liver, and kidney preserved en bloc or as a single preparation. In addition, DADLE has been recently demonstrated to promote survival of neurons in the central nervous system. Exposure to DADLE dose-dependently enhanced cell viability of cultured primary rat fetal dopaminergic cells. Subsequent transplantation of these DADLE-treated dopaminergic cells into the Parkinsonian rat brain resulted in a two-fold increase in surviving grafted cells. Interestingly, delivery of DADLE alone protected against dopaminergic depletion in a rodent model of Parkinson s disease. Similarly, DADLE blocked and reversed the dopaminergic terminal damage induced by methamphetamine (METH). Such neuroprotective effects of DADLE against METH neurotoxicity was accompanied by attenuation of mRNA expressions of a tumor necrosis factor p53 and an immediate early gene c-fos. In parallel to these beneficial effects of DADLE on the dopaminergic system, DADLE also ameliorated the neuronal damage induced by ischemia-reperfusion following a transient middle cerebral artery occlusion. In vitro replication of this ischemia cell death by serum-deprivation of PC12 cells revealed that DADLE exerted neuroprotection in a naltrexone-sensitive manner. These results taken together suggest that DADLE stands as a novel therapeutic agent. In this review paper, we present laboratory evidence supporting the use of DADLE for protection of peripheral and central nervous system.

Effect of opioid receptor ligands on the μ-S196A knock-in and μ knockout mouse vas deferens

We have determined the effect of naltrexone, naloxone, [ d-Ala 2, d-Leu 5]enkephalin (DADLE), and morphine on the μ-S196A opioid receptor knock-in and μ-opioid receptor knockout mouse vas deferens preparations. The antagonists, naltrexone and naloxone, exhibited agonist activity and possessed IC 50 values that were 14- and 37-fold greater than morphine on the S196A preparation. Morphine was found to be threefold more potent at S196A relative to wild-type μ-opioid receptor. The mouse vas deferens data suggest that S196 in transmembrane helix 4 of the μ-opioid receptor modulates efficacy. It is proposed that this may be due to decreased dimerization of the receptor. Identical IC 50 values of DADLE obtained on the wild-type, S196A knock-in, and μ-opioid receptor knockout preparations support the absence of μ–δ heterodimers in the mouse vas deferens.

Purification and mass spectrometric analysis of the μ opioid receptor

A mouse μ opioid receptor was engineered to contain a FLAG epitope at the amino-terminus and a hexahistidine tag at the carboxyl-terminus to facilitate purification. Selection of transfected human embryonic kidney (HEK) 293 cells yielded a cell line that expressed the receptor with a B max of 10 pmol/mg protein. 3[H]Bremazocine exhibited high affinity binding to the epitope-tagged μ opioid receptor with a K D of 1.0 nM. The agonists [ d-Ala 2, N-Me-Phe 4,Gly 5-ol]enkephalin (DAMGO), morphine and [ d-Ala 2, d-Leu 5]enkephalin (DADL) competitively inhibited bremazocine binding to the tagged μ receptor with K I's of 3.5, 17 and 70 nM, respectively. Chronic treatment of cells expressing the epitope-tagged μ receptor with DAMGO resulted in down-regulation of the receptor, indicating that the tagged receptor retained the capacity to mediate signal transduction. The μ receptor was solubilized from HEK 293 cell membranes with n-dodecyl-β- d-maltoside in an active form that maintained high affinity bremazocine binding. Sequential use of wheat germ agglutinin (WGA)–agarose chromatography, Sephacryl S300 gel filtration chromatography, immobilized metal affinity chromatography, immunoaffinity chromatography, and sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS/PAGE) permitted purification of the receptor. The purified μ opioid receptor was a glycoprotein that migrated on SDS/PAGE with an apparent molecular mass of 80 kDa. Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry was used to identify and characterize peptides derived from the μ opioid receptor following in-gel digestion with trypsin or chymotrypsin, and precursor-derived tandem mass spectrometry (ms/ms) confirmed the identity of several peptides derived from enzymatic digestion of the μ opioid receptor.

D-Ala 2, d-Leu 5] enkephalin (DADLE) protects liver against ischemia-reperfusion injury in the rat

Background [ d-Ala 2, d-Leu 5] enkephalin (DADLE) is a synthetic delta class of opioid and is reported to induce hibernation as well as hibernation induction trigger (HIT) in the serum of hibernating mammals. DADLE and HIT have been demonstrated to protect the heart, lung, and jejunum against ischemia-reperfusion (I-R) injury. In the present study, we examined the effect of DADLE on I-R injury of the liver in rats. Materials and methods After administration of DADLE (DADLE group) or normal saline as a vehicle (Control group), partial hepatic ischemia was induced by occluding the vessels supplying 92% of the liver for 45 min, followed by declamping the vessels and resection of the non-ischemic lobe. After 120 min of reperfusion, serum glutamic-pyruvic transaminase (GPT), hyaluronic acid (HA) levels, and concentrations of malondialdehyde (MDA) of the liver tissue were measured. Additionally, bile output from the ischemic lobes was measured after reperfusion. Results GPT levels were significantly lower in the DADLE group as compared to those of the Control group ( P < 0.05), but the serum levels of HA were not different between the two groups. The concentrations of MDA of the liver tissue were significantly lower in the DADLE group than in the Control group ( P < 0.01). The bile output after reperfusion was not significantly different between the two groups. Conclusion DADLE protects against I-R injury in hepatocytes, but not in the sinusoidal endothelial cells of the liver in rats. An anti-oxidative effect is suggested to be responsible for this effect.

Peripheral opioidergic regulation of the tracheobronchial mucociliary transport system.

We hypothesized that, in the airway mucosa, opioids are inhibitory neural modulators that cause an increase in net water absorption in the airway mucosa (as in the gut). Changes in bidirectional water fluxes across ovine tracheal mucosa in response to basolateral application of the opioid peptides beta-endorphin, dynorphin A-(1-8), and [d-Ala(2), d-Leu(5)]-enkephalin (DADLE) were measured. beta-Endorphin and dynorphin A-(1-8) decreased luminal-to-basolateral water fluxes, and dynorphin A-(1-8) and DADLE increased basolateral-to-luminal water flux. These responses were electroneutral. In seven beagle dogs, administration of aerosolized beta-endorphin (1 mg) to the tracheobronchial airways decreased the clearance of radiotagged particles from the bronchi in 1 h from 34.7 to 22.0% (P < 0.001). Naloxone abrogated the beta-endorphin-induced changes in vitro and in vivo. Contrary to our hypothesis, the opioid-induced changes in water fluxes would all lead to a predictable increase in airway surface fluid. The beta-endorphin-induced increases in airway fluid together with reduced bronchial mucociliary clearance may produce procongestive responses when opioids are administered as antitussives.

Chronic [ d-Ala 2, d-Leu 5]enkephalin treatment increases the nerve growth factor in adult mouse brain

The δ opioid peptide [ d-Ala 2, d-Leu 5]enkephalin (DADLE) has been shown to enhance the survival of dopaminergic neurons. Here, we found that chronic treatment with DADLE caused a significant increase in nerve growth factor (NGF) in the hippocampus and the midbrain of adult albino Swiss (CD-1) mice, but not in the striatum or frontal cortex. Glia-derived neurotrophic factor (GDNF) was not significantly affected. Thus, the neuroprotective action of DADLE may be mediated in part by NGF.

Chronic Morphine Treatment Inhibits Opioid Receptor Desensitization and Internalization

Chronic opioid receptor (OR) activation by morphine causes distinct cellular adaptations responsible for the development of tolerance. The present study examines the effect of chronic morphine exposure on the ability of high-efficacy agonists to mediate delta-OR (DOR) and mu-OR (MOR) uncoupling and internalization, two regulatory mechanisms contributing to rapid desensitization of OR function. Chronic morphine treatment (1 microm; 72 hr) of DOR carrying neuroblastoma x glioma (NG108-15) hybrid cells, a prototypical model system frequently used to study cellular aspects of opioid tolerance, completely blocked the capacity of [d-Ala2, d-Leu5]enkephalin (DADLE) and etorphine to desensitize opioid-stimulated [35S]GTPgammaS binding and to mediate DOR internalization. Similar findings were obtained on stably DOR- and MOR-transfected human embryonic kidney (HEK) 293 cells. Chronic morphine treatment also heterologously impaired agonist regulation of non-opioid G-protein-coupled receptors, such as the m(4)-muscarinic acetylcholine receptor and the brain-type cannabinoid receptor. As a possible underlying mechanism, we found that chronic morphine treatment completely blocked agonist-induced redistribution of beta-arrestin1 in both NG108-15 and stably MOR-transfected HEK293 cells. Moreover, attenuation of beta-arrestin1 function appears to depend on persistent stimulation of MAP kinase activity during the course of chronic morphine treatment, because coincubation of the cells together with the MAP kinase blocker PD98059 fully restored beta-arrestin1 translocation and receptor internalization. These results demonstrate that chronic morphine treatment produces adaptational changes at the beta-arrestin1 level, which in turn attenuates agonist-mediated desensitization and internalization of G-protein-coupled receptors.