[D-Ala2, D-Leu5]-enkephalin (DADLE) provides protection against myocardial ischemia reperfusion injury by inhibiting Wnt/ß-Catenin pathway.

BACKGROUND: Acute myocardial infarction is one of the leading causes of death worldwide. Myocardial ischemia reperfusion (MI/R) injury occurs immediately after the coronary reperfusion and aggravates myocardial ischemia. Whether the Wnt/ß-Catenin pathway is involved in the protection against MI/R injury by DADLE has not been evaluated. Therefore, the present study aimed to investigate the protective effect of DADLE against MI/R injury in a mouse model and to further explore the association between DADLE and the Wnt/ß-Catenin pathway. METHODS: Forty-four mice were randomly allocated to four groups: Group Control (PBS Control), Group D 0.25 (DADLE 0.25 mg/kg), Group D 0.5 (DADLE 0.5 mg/kg), and Group Sham. In the control and DADLE groups, myocardial ischemia injury was induced by occluding the left anterior descending coronary artery (LAD) for 45 min. PBS and DADLE were administrated, respectively, 5 min before reperfusion. The sham group did not go through LAD occlusion. 24 h after reperfusion, functions of the left ventricle were assessed through echocardiography. Myocardial injury was evaluated using TTC double-staining and HE staining. Levels of myocardial enzymes, including CK-MB and LDH, in the serum were determined using ELISA kits. Expression of caspase-3, TCF4, Wnt3a, and ß-Catenin was evaluated using the Western blot assay. RESULTS: The infarct area was significantly smaller in the DADLE groups than in the control group (P < 0.01). The histopathology score and serum levels of myocardial enzymes were significantly lower in the DADLE groups than in the control group (P < 0.01). DADLE significantly improved functions of the left ventricle (P < 0.01), decreased expression of caspase-3 (P < 0.01), TCF4 (P < 0.01), Wnt3a (P < 0.05), and ß-Catenin (P < 0.01) compared with PBS. CONCLUSIONS: The present study showed that DADLE protected the myocardium from MI/R through suppressing the expression of caspase-3, TCF4, Wnt3a, and ß-Catenin and consequently improving functions of the left ventricle in I/R model mice. The TCF4/Wnt/ß-Catenin signaling pathway might become a therapeutic target for MI/R treatment.

DADLE promotes motor function recovery by inhibiting cytosolic phospholipase A2 mediated lysosomal membrane permeabilization after spinal cord injury.

BACKGROUND AND PURPOSE: Autophagy is a protective factor for controlling neuronal damage, while necroptosis promotes neuroinflammation after spinal cord injury (SCI). DADLE (D-Ala2 , D-Leu5 ]-enkephalin) is a selective agonist for delta (δ) opioid receptor and has been identified as a promising drug for neuroprotection. The aim of this study was to investigate the mechanism/s by which DADLE causes locomotor recovery following SCI. EXPERIMENTAL APPROACH: Spinal cord contusion model was used and DADLE was given by i.p. (16 mg·kg-1 ) in mice for following experiments. Motor function was assessed by footprint and Basso mouse scale (BMS) score analysis. Western blotting used to evaluate related protein expression. Immunofluorescence showed the protein expression in each cell and its distribution. Network pharmacology analysis was used to find the related signalling pathways. KEY RESULTS: DADLE promoted functional recovery after SCI. In SCI model of mice, DADLE significantly increased autophagic flux and inhibited necroptosis. Concurrently, DADLE restored autophagic flux by decreasing lysosomal membrane permeabilization (LMP). Additionally, chloroquine administration reversed the protective effect of DADLE to inhibit necroptosis. Further analysis showed that DADLE decreased phosphorylated cPLA2 , overexpression of cPLA2 partially reversed DADLE inhibitory effect on LMP and necroptosis, as well as the promotion autophagy. Finally, AMPK/SIRT1/p38 pathway regulating cPLA2 is involved in the action DADLE on SCI and naltrindole inhibited DADLE action on δ receptor and on AMPK signalling pathway. CONCLUSION AND IMPLICATION: DADLE causes its neuroprotective effects on SCI by promoting autophagic flux and inhibiting necroptosis by decreasing LMP via activating δ receptor/AMPK/SIRT1/p38/cPLA2 pathway.

Effect of dalargin on apoptosis of l929 fibroblasts during cold stress.

BACKGROUND: The search for compounds that can prevent cold stress-attributed apoptosis is of immediate interest. In this regard, the use of neuropeptides, in particular synthetic leu-enkephalin, as protectors is promising, due to their ability to prevent the development of apoptosis under some stresses. OBJECTIVE: To study apoptotic phenomena after cold stress and to evaluate the protective effect of dalargin on these processes. MATERIALS AND METHODS: The study was performed on a L929 fibroblast line. The impact of cold stress and the protective effect of dalargin on apoptosis against cold stress were evaluated using morphological parameters, distortion of cell membrane asymmetry and release of cytochrome C into the cell cytoplasm. To assess the proliferative potential of fibroblasts, mechanical damage to the monolayer was modeled as a scratch wound. RESULTS: The study showed that cold stress induced apoptosis in L929 fibroblasts and reduced proliferation in the fibroblast monolayers. Conspicuous apoptotic changes were found to develop only after a certain time after cold exposure, when the cells were returned to normothermia. Dalargin was demonstrated to exert a protective effect on proliferation and against apoptosis during cold stress. Using the opioid receptor antagonist naloxone, we revealed that the protective mechanism of dalargin appeared to be due to activation of delta-opioid receptors of L929 fibroblasts, which affected the development of apoptosis. CONCLUSION: In addition to their fundamental value, these findings are of practical importance since neuropeptides, in particular dalargin, added to perfusion solutions and media for hypothermic preservation of organs and cells, can improve their efficiency. Doi.org/10.54680/fr23610110212.

Activation of δ-opioid Receptors in Anterior Cingulate Cortex Alleviates Affective Pain in Rats.

The negative emotions caused by persistent pain, called affective pain, are known to seriously affect human physical and mental health. The anterior cingulate cortex (ACC), especially the rostral ACC (rACC) plays a key role in the development of this affective pain. N-methyl-d-aspartate (NMDA) receptors, which are widely distributed in the ACC, are involved in the regulation of emotional behavior. It is well known that activation of opioid receptors can relieve pain, but whether it can alleviate affective pain is not clear. In the present study, conditioned place avoidance (CPA) responses induced by complete Freund's adjuvant (CFA) were used to represent the affective pain of place aversion. The behavioral measurements were synchronously combined with multichannel electrophysiological recordings of the discharge frequency of rACC pyramidal neurons to explore whether affective pain could be alleviated by the synthetic opioid [D-Ala2, D-Leu5]-Enkefalin (DADLE), an agonist of δ-opioid receptors. To further investigate this treatment as a mechanism for the relief of affective pain in CFA-treated animals, we used whole-cell patch recordings in slice preparations of the rACC region to determine the dose-dependent effects of DADLE on NMDA receptor-mediated currents. Then, western blot was used to determine levels of phosphorylated NMDA receptor subunits GluN1, GluN2 and GluN3 as affected by the δ-opioid receptor activation. The results showed that activation of δ-opioid receptors down-regulates the phosphorylation of NMDA receptor subunits, thereby inhibiting NMDA currents, decreasing the discharge frequency of rACC pyramidal neurons, and reversing the CPA response. Thus, δ-opioid receptor activation in the rACC region can alleviate affective pain.

Effect of Leu-Enkephalin Analogue on the Myeloid Compartment of the Blood System in Hypothyroid White Rats under Stress Conditions.

In white rats with experimental hypothyroidism, changes in the myeloid compartment of the blood system induced by 6-h immobilization stress and the corrective effect of the analogue of leu-enkephalin (dalargin) on these shifts was analyzed. It was found that in rats with hypothyroidism, stress in the anxiety stage did not cause leukocytosis typical of euthyroid animals, but at the stage of resistance provoked leukopenia at the expense of eosinopenia and neutropenia with depletion of the intramedullary reserve. Dalargin increased white blood cells count, neutrophil count, and the intramedullary depot of these cells.

Lysozyme-dalargin self-organization at the aqueous-air and liquid-liquid interfaces.

An experimental study of protein-peptide binding was performed by means of radiochemical and spectroscopic methods. Lysozyme and dalargin were chosen due to their biological and physiological importance. By means of tensiometry and radiochemical assays, it was found that dalargin possesses rather high surface activity at the aqueous-air and aqueous-p-xylene interfaces to be substituted by protein. Dalargin forms a hydrophobic complex with lysozyme in which the secondary structure of lysozyme is preserved. When lysozyme forms a mixed adsorption layer with dalargin at the aqueous-air surface, the peptide prevents protein from concentrating in the subsurface monolayer. In the presence of p-xylene protein in the interface, reorganization occurs quickly, so there is no lag in the interfacial tension time dependence. The interfacial tension in this case is controlled by protein and/or protein-peptide complexes. An increase in the enzymatic activity of lysozyme in the presence of dalargin was confirmed by a docking model that suggests the formation of hydrogen bonds between dalargin and amino acid residues in the active site.

Activation of the δ opioid receptor relieves cerebral ischemic injury in rats via EGFR transactivation.

Delta opioids are thought to relieve ischemic injury and have tissue-protective properties. However, the detailed mechanisms of delta opioids have not been well identified. Receptor tyrosine kinases (RTKs), such as epidermal growth factor receptor (EGFR), have been shown to mediate downstream signals of δ opioid receptor (δOR) activation through the metalloproteinase (MMP)-dependent EGF-like growth factor (HB-EGF) excretion pathway, which is called transactivation. In this study, to investigate the role of EGFR in δOR-induced anti-ischemic effects in the brain, we applied the middle cerebral artery occlusion (MCAO) model followed by reperfusion to mimic ischemic stroke injury in rats. Pre-treatment with the δOR agonist [D-ala2, D-leu5] enkephalin (DADLE) improved the neurologic deficits and the decreased infarct volume caused by cerebral ischemia/reperfusion injury, which were blocked by the EGFR inhibitor AG1478 and the MMP inhibitor GM6001, respectively. Further results indicated that DADLE activated EGFR, Akt and ERK1/2 and upregulated EGFR expression in the hippocampus in a time-dependent manner, which were inhibited by AG1478 and GM6001. The enzyme-linked immunosorbent assay (ELISA) results showed that δOR activation led to an increase in HB-EGF release, but HB-EGF in tissue was downregulated at the mRNA and protein levels. Moreover, this protective action caused by δOR agonists may involve attenuated hippocampal cellular apoptosis. Overall, these results demonstrate that MMP-mediated transactivation of EGFR is essential for δOR agonist-induced MCAO/reperfusion injury relief. These findings provide a potential molecular mechanism for the neuroprotective property of δOR and may add new insight into mitigating or preventing injury.

Effects of the Delta Opioid Receptor Agonist DADLE in a Novel Hypoxia-Reoxygenation Model on Human and Rat-Engineered Heart Tissue: A Pilot Study.

Intermittent hypoxia and various pharmacological compounds protect the heart from ischemia reperfusion injury in experimental approaches, but the translation into clinical trials has largely failed. One reason may lie in species differences and the lack of suitable human in vitro models to test for ischemia/reperfusion. We aimed to develop a novel hypoxia-reoxygenation model based on three-dimensional, spontaneously beating and work performing engineered heart tissue (EHT) from rat and human cardiomyocytes. Contractile force, the most important cardiac performance parameter, served as an integrated outcome measure. EHTs from neonatal rat cardiomyocytes were subjected to 90 min of hypoxia which led to cardiomyocyte apoptosis as revealed by caspase 3-staining, increased troponin I release (time control vs. 24 h after hypoxia: cTnI 2.7 vs. 6.3 ng/mL, ** p = 0.002) and decreased contractile force (64 ± 6% of baseline) in the long-term follow-up. The detrimental effects were attenuated by preceding the long-term hypoxia with three cycles of 10 min hypoxia (i.e., hypoxic preconditioning). Similarly, [d-Ala2, d-Leu5]-enkephalin (DADLE) reduced the effect of hypoxia on force (recovery to 78 ± 5% of baseline with DADLE preconditioning vs. 57 ± 5% without, p = 0.012), apoptosis and cardiomyocyte stress. Human EHTs presented a comparable hypoxia-induced reduction in force (55 ± 5% of baseline), but DADLE failed to precondition them, likely due to the absence of δ-opioid receptors. In summary, this hypoxia-reoxygenation in vitro model displays cellular damage and the decline of contractile function after hypoxia allows the investigation of preconditioning strategies and will therefore help us to understand the discrepancy between successful conditioning in vitro experiments and its failure in clinical trials.

Endosomal signaling of delta opioid receptors is an endogenous mechanism and therapeutic target for relief from inflammatory pain.

Whether G protein-coupled receptors signal from endosomes to control important pathophysiological processes and are therapeutic targets is uncertain. We report that opioids from the inflamed colon activate δ-opioid receptors (DOPr) in endosomes of nociceptors. Biopsy samples of inflamed colonic mucosa from patients and mice with colitis released opioids that activated DOPr on nociceptors to cause a sustained decrease in excitability. DOPr agonists inhibited mechanically sensitive colonic nociceptors. DOPr endocytosis and endosomal signaling by protein kinase C (PKC) and extracellular signal-regulated kinase (ERK) pathways mediated the sustained inhibitory actions of endogenous opioids and DOPr agonists. DOPr agonists stimulated the recruitment of Gαi/o and ß-arrestin1/2 to endosomes. Analysis of compartmentalized signaling revealed a requirement of DOPr endocytosis for activation of PKC at the plasma membrane and in the cytosol and ERK in the nucleus. We explored a nanoparticle delivery strategy to evaluate whether endosomal DOPr might be a therapeutic target for pain. The DOPr agonist DADLE was coupled to a liposome shell for targeting DOPr-positive nociceptors and incorporated into a mesoporous silica core for release in the acidic and reducing endosomal environment. Nanoparticles activated DOPr at the plasma membrane, were preferentially endocytosed by DOPr-expressing cells, and were delivered to DOPr-positive early endosomes. Nanoparticles caused a long-lasting activation of DOPr in endosomes, which provided sustained inhibition of nociceptor excitability and relief from inflammatory pain. Conversely, nanoparticles containing a DOPr antagonist abolished the sustained inhibitory effects of DADLE. Thus, DOPr in endosomes is an endogenous mechanism and a therapeutic target for relief from chronic inflammatory pain.

New Combined Eye Gel with Alpha-2-Beta Interferon.

The results of the development of combined eye gel with interferon alpha-2-beta are presented. Experimental samples of the gel based on different gelling agents were prepared and their biotechnological and technological characteristics (the absence of the cytotoxic effect, aggregation stability, osmotic activity, bioadhesion, and rheological parameters) were evaluated. The composition with hydroxyethyl cellulose, Natrosol 250HHX, in a concentration of 1.5% as a gelling agent showed the best results and the best one-year stability.

Effects of D­Ala2, D­Leu5­Enkephalin pre­ and post­conditioning in a rabbit model of spinal cord ischemia and reperfusion injury.

It has recently been revealed that during the aorta­clamped period, D­Ala2, D­Leu5­Enkephalin (DADLE) infusion can protect the spinal cord against ischemia and reperfusion (I/R) injury. However, the protective effects of DADLE administration prior to ischemia or at the time of early reperfusion have not yet been investigated. Drug pre­ or post­conditioning can serve as a more valuable clinical strategy. Therefore, the present study was designed to investigate the neuroprotective effect of DADLE infusion at different time intervals in order to determine the optimum time point for ischemic spinal cord protection. A total of 40 New Zealand white rabbits were randomly divided into 5 groups: Sham­operated (Sham), normal saline pre­conditioning (NS), DADLE per­conditioning (Dper), DADLE pre­conditioning (Dpre) and DADLE post­conditioning (Dpost). All animals were subjected to spinal cord ischemia for 30 min followed by 48 h reperfusion. Hind limb motor functions were assessed according to the Tarlov criterion when the animals regained consciousness, 6, 24 and 48 h after reperfusion. Histological analysis and the number of viable α­motor neurons were also used to assess the extent of spinal cord injury. Compared with the NS group, the Tarlov scores and the number of normal neurons were significantly higher in the Dper group (P<0.05), which were consistent with the results of a previous study. In addition, the paraplegia rate and loss of normal motor neurons were lower in the DADLE per­ and post­conditioning groups compared with the DADLE pre­conditioning; however, these were not statistically significant. DADLE 0.05 mg/kg administration at three time points all mitigated normal motor neuron injury in the anterior horn and decreased the paraplegia rates in rabbits. The therapeutic benefits appeared best in the post­conditioning group with DADLE, and worst in the pre­conditioning group.

Activation of HIF-1α by δ-Opioid Receptors Induces COX-2 Expression in Breast Cancer Cells and Leads to Paracrine Activation of Vascular Endothelial Cells.

Opioids promote tumor angiogenesis in mammary malignancies, but the underlying signaling mechanism is largely unknown. The current study investigated the hypothesis that stimulation of δ-opioid receptors (DOR) in breast cancer (BCa) cells activates the hypoxia-inducible factor 1α (HIF-1α), which triggers synthesis and release of diverse angiogenic factors. Immunoblotting revealed that incubation of human MCF-7 and T47D breast cancer cells with the DOR agonist d-Ala2,d-Leu5-enkephalin (DADLE) resulted in a transient accumulation and thus activation of HIF-1α DADLE-induced HIF-1α activation preceded PI3K/Akt stimulation and was blocked by the DOR antagonist naltrindole and naloxone, pertussis toxin, different phosphoinositide 3-kinase (PI3K) inhibitors, and the Akt inhibitor Akti-1/2. Whereas DADLE exposure had no effect on the expression and secretion of vascular endothelial growth factor (VEGF) in BCa cells, an increased abundance of cyclooxygenase-2 (COX-2) and release of prostaglandin E2 (PGE2) was detected. DADLE-induced COX-2 expression was also observed in three-dimensional cultured MCF-7 cells and impaired by PI3K/Akt inhibitors and the HIF-1α inhibitor echinomycin. Supernatant from DADLE-treated MCF-7 cells triggered sprouting of endothelial (END) cells, which was blocked when MCF-7 cells were pretreated with echinomycin or the COX-2 inhibitor celecoxib. Also no sprouting was observed when END cells were exposed to the PGE2 receptor antagonist PF-04418948. The findings together indicate that DOR stimulation in BCa cells leads to PI3K/Akt-dependent HIF-1α activation and COX-2 expression, which trigger END cell sprouting by paracrine activation of PGE2 receptors. These findings provide a potential mechanism of opioid-driven tumor angiogenesis and thus therapeutic targets to combat the tumor-angiogenic opioid effect. SIGNIFICANCE STATEMENT: Opioids are indispensable analgesics for treating cancer-related pain. However, opioids were found to promote tumor growth and metastasis, which questions the use of these potent pain-relieving drugs in cancer patients. Enhanced tumor vascularization after opioid treatment implies that tumor progression results from angiogenic opioid effects. Thus, understanding the signaling mechanism of opioid-driven tumor angiogenesis helps to identify therapeutic targets to combat these undesired tumor effects. The present study reveals that stimulation of δ-opioid receptors in breast cancer cells leads to an activation of HIF-1α and expression of COX-2 via PI3K/Akt stimulation, which results in a paracrine activation of vascular endothelial cells by prostaglandin E2 receptors.

Role of RGS12 in the differential regulation of kappa opioid receptor-dependent signaling and behavior.

Kappa opioid receptor (KOR) agonists show promise in ameliorating disorders, such as addiction and chronic pain, but are limited by dysphoric and aversive side effects. Clinically beneficial effects of KOR agonists (e.g., analgesia) are predominantly mediated by heterotrimeric G protein signaling, whereas ß-arrestin signaling is considered central to their detrimental side effects (e.g., dysphoria/aversion). Here we show that Regulator of G protein Signaling-12 (RGS12), via independent signaling mechanisms, simultaneously attenuates G protein signaling and augments ß-arrestin signaling downstream of KOR, exhibiting considerable selectivity in its actions for KOR over other opioid receptors. We previously reported that RGS12-null mice exhibit increased dopamine transporter-mediated dopamine (DA) uptake in the ventral (vSTR), but not dorsal striatum (dSTR), as well as reduced psychostimulant-induced hyperlocomotion; in the current study, we found that these phenotypes are reversed following KOR antagonism. Fast-scan cyclic voltammetry studies of dopamine (DA) release and reuptake suggest that striatal disruptions to KOR-dependent DAergic neurotransmission in RGS12-null mice are restricted to the nucleus accumbens. In both ventral striatal tissue and transfected cells, RGS12 and KOR are seen to interact within a protein complex. Ventral striatal-specific increases in KOR levels and KOR-induced G protein activation are seen in RGS12-null mice, as well as enhanced sensitivity to KOR agonist-induced hypolocomotion and analgesia-G protein signaling-dependent behaviors; a ventral striatal-specific increase in KOR levels was also observed in ß-arrestin-2-deficient mice, highlighting the importance of ß-arrestin signaling to establishing steady-state KOR levels in this particular brain region. Conversely, RGS12-null mice exhibited attenuated KOR-induced conditioned place aversion (considered a ß-arrestin signaling-dependent behavior), consistent with the augmented KOR-mediated ß-arrestin signaling seen upon RGS12 over-expression. Collectively, our findings highlight a role for RGS12 as a novel, differential regulator of both G protein-dependent and -independent signaling downstream of KOR activation.

[D-Ala2, D-Leu5] Enkephalin Improves Liver Preservation During Normothermic Ex Vivo Perfusion.

BACKGROUND: Meeting the metabolic demands of donor livers using normothermic ex vivo liver perfusion (NEVLP) preservation technology is challenging. The delta opioid agonist [D-Ala2, D-Leu5] enkephalin (DADLE) has been reported to decrease the metabolic demand in models of ischemia and cold preservation. We evaluated the therapeutic potential of DADLE by investigating its ability to protect against oxidative stress and hepatic injury during normothermic perfusion. MATERIALS AND METHODS: Primary rat hepatocytes were used in an in vitro model of oxidative stress to determine the minimum dose of DADLE needed to induce protection and the mechanisms associated with protection. NEVLP was then used to induce injury in rat livers and determine the effectiveness of DADLE in preventing liver injury. RESULTS: In hepatocytes, DADLE was protective against oxidative stress and led to a decrease in phosphorylation of JNK and p38. Naltrindole, a δ-opioid receptor antagonist, blocked this effect. DADLE also activated the PI3K/Akt signaling pathway, and PI3K/Akt inhibition decreased the protective effects of DADLE treatment. In addition, DADLE treatment during NEVLP resulted in lower perfusate alanine aminotransferase and tissue malondialdehyde and better tissue adenosine triphosphate and glutathione. Furthermore, perfusion with DADLE compared with perfusate alone preserved tissue architecture. CONCLUSIONS: DADLE confers protection against oxidative stress in hepatocytes and during NEVLP. These data suggest that the mechanism of protection involved the prevention of mitochondrial dysfunction by opioid receptor signaling and subsequent increased expression of prosurvival/antiapoptotic signaling pathways. Altogether, data suggest that opioid receptor agonism may serve as therapeutic target for improved liver protection during NEVLP.

Delta Opioid Peptide [d-Ala2, d-Leu5] Enkephalin (DADLE) Exerts a Cytoprotective Effect in Astrocytes Exposed to Oxygen-Glucose Deprivation by Inducing Autophagy.

Astrocytes protection and functional regulation are important strategies to protect against neuronal damage caused by ischemia. Activation of the delta opioid receptor (DOR) could reduce astrocytes damage, although the mechanism remains unclear. The present study aimed to test the effect of DOR activation on autophagy in astrocytes exposed to oxygen-glucose deprivation (OGD), and to further investigate whether this effect has a protective effect on astrocytes. Primary cultured rat cortical astrocytes were treated with various doses of [d-Ala2, d-Leu5]-Enkephalin (DADLE, a selective DOR agonist) followed by 6 h OGD. Cell viability was evaluated by CCK-8 assay and lactate dehydrogenase release. Autophagic vacuole was analyzed with LC3 immunofluorescent staining. The levels of autophagy and apoptosis-related proteins were analyzed by western blot. Results demonstrated that treatment with 10 nM DADLE was sufficient to increase cell viability and induced autophagy in astrocytes. The DADLE-induced autophagy displayed a cytoprotective effect on astrocytes. Inhibition of autophagy by 3-methyladenine (3-MA, an autophagy inhibitor) reversed the protective effect of DADLE. Naltrindole (a DOR antagonist) only partially antagonized the role of DADLE, which indicated that DADLE might have a cytoprotective mechanism independent of DOR. Further results showed that DADLE significantly enhanced the level of Bcl-2 protein and reduced the level of Bax protein in astrocytes exposed to OGD. Our results suggest a novel mechanism in which DADLE induces autophagy in astrocytes and exerts cytoprotective effects by inhibiting apoptosis.

Inflammation-associated changes in DOR expression and function in the mouse colon.

Endogenous opioids activate opioid receptors (ORs) in the enteric nervous system to control intestinal motility and secretion. The µ-OR mediates the deleterious side effects of opioid analgesics, including constipation, respiratory depression, and addiction. Although the δ-OR (DOR) is a promising target for analgesia, the function and regulation of DOR in the colon are poorly understood. This study provides evidence that endogenous opioids activate DOR in myenteric neurons that may regulate colonic motility. The DOR agonists DADLE, deltorphin II, and SNC80 inhibited electrically evoked contractions and induced neurogenic contractions in the mouse colon. Electrical, chemical, and mechanical stimulation of the colon evoked the release of endogenous opioids, which stimulated endocytosis of DOR in the soma and proximal neurites of myenteric neurons of transgenic mice expressing DOR fused to enhanced green fluorescent protein. In contrast, DOR was not internalized in nerve fibers within the circular muscle. Administration of dextran sulfate sodium induced acute colitis, which was accompanied by DOR endocytosis and an increased density of DOR-positive nerve fibers within the circular muscle. The potency with which SNC80 inhibited neurogenic contractions was significantly enhanced in the inflamed colon. This study demonstrates that DOR-expressing neurons in the mouse colon can be activated by exogenous and endogenous opioids. Activated DOR traffics to endosomes and inhibits neurogenic motility of the colon. DOR signaling is enhanced during intestinal inflammation. This study demonstrates functional expression of DOR by myenteric neurons and supports the therapeutic targeting of DOR in the enteric nervous system. NEW & NOTEWORTHY DOR is activated during physiologically relevant reflex stimulation. Agonist-evoked DOR endocytosis is spatially and temporally regulated. A significant proportion of DOR is internalized in myenteric neurons during inflammation. The relative proportion of all myenteric neurons that expressed DOR and the overlap with the nNOS-positive population are increased in inflammation. DOR-specific innervation of the circular muscle is increased in inflammation, and this is consistent with enhanced responsiveness to the DOR agonist SNC80.

A Genetically Encoded Biosensor Reveals Location Bias of Opioid Drug Action.

Opioid receptors (ORs) precisely modulate behavior when activated by native peptide ligands but distort behaviors to produce pathology when activated by non-peptide drugs. A fundamental question is how drugs differ from peptides in their actions on target neurons. Here, we show that drugs differ in the subcellular location at which they activate ORs. We develop a genetically encoded biosensor that directly detects ligand-induced activation of ORs and uncover a real-time map of the spatiotemporal organization of OR activation in living neurons. Peptide agonists produce a characteristic activation pattern initiated in the plasma membrane and propagating to endosomes after receptor internalization. Drugs produce a different activation pattern by additionally driving OR activation in the somatic Golgi apparatus and Golgi elements extending throughout the dendritic arbor. These results establish an approach to probe the cellular basis of neuromodulation and reveal that drugs distort the spatiotemporal landscape of neuronal OR activation.

Prolonged DADLE exposure epigenetically promotes Bcl-2 expression and elicits neuroprotection in primary rat cortical neurons via the PI3K/Akt/NF-κB pathway.

Both in vivo and in vitro studies have shown the beneficial effects of the delta-opioid receptor (DOR) on neurodegeneration in hypoxia/ischemia. We previously reported that DOR stimulation with [(D-Ala2, D-Leu5) enkephalin] (DADLE), a potent DOR agonist, for both a short (minutes) and long (days) time has notable protective effects against sodium azide (NaN3)-induced cell injury in primary cultured rat cortical neurons. We further demonstrated that short-term DADLE stimulation increased neuronal survival through the PKC-mitochondrial ERK pathway. However, the mechanisms underlying long-term neuroprotection by DADLE remain unclear. Here, we showed that DOR stimulation with DADLE (0.1 µmol/L) for 2 d selectively activates the PI3K/Akt/NF-κB pathway in NaN3-treated neurons; this activation increased Bcl-2 expression, attenuated Cyto c release and promoted neuronal survival. Further investigation revealed that sustained DADLE stimulation increased Bcl-2 expression by enhancing NF-κB binding to the Bcl-2 promoter and upregulating the histone acetylation levels of the Bcl-2 promoter. Our results demonstrate that prolonged DADLE exposure epigenetically promotes Bcl-2 expression and elicits neuroprotective effects in the NaN3 model via the PI3K/Akt/NF-κB pathway.

The Delta Opioid Peptide DADLE Represses Hypoxia-Reperfusion Mimicked Stress Mediated Apoptotic Cell Death in Human Mesenchymal Stem Cells in Part by Downregulating the Unfolded Protein Response and ROS along with Enhanced Anti-Inflammatory Effect.

Hypoxia-reperfusion (H/R) emblems a plethora of pathological conditions which is potent in contributing to the adversities encountered by human mesenchymal stem cells (hMSCs) in post-transplant microenvironment, resulting in transplant failure. D-Alanine 2, Leucine 5 Enkephaline (DADLE)-mediated delta opioid receptor (DOR) activation is well-known for its recuperative properties in different cell types like neuronal and cardiomyocytes. In the current study its effectiveness in assuaging hMSC mortality under H/R-like insult has been delineated. The CoCl2 mimicked H/R conditions in vitro was investigated upon DOR activation, mediated via DADLE. hMSCs loss of viability, reactive oxygen species (ROS) production, inflammatory responses and disconcerted unfolded protein response (UPR) were assessed using AnnexinV/PI flow cytometry, fluorescence imaging, mitochondrial complex 1 assay, quantitative PCR, immunoblot analysis and ELISA. H/R like stress induced apoptosis of hMSCs was significantly mitigated by DADLE via modulation of the apoptotic regulators (Bcl-2/Bax) along with significant curtailment of ROS and mitochondrial complex 1 activity. DADLE concomitantly repressed the misfolded protein aggregation, alongside the major UPR sensors: PERK/BiP/IRE-1α /ATF-6, evoked due to the H/R mimicked endoplasmic reticulum stress. Undermined phosphorylation of the Akt signalling pathway was observed, which concerted its effect onto regulating both the pro and anti-inflammatory cytokines, actuated as a response to the H/R-like insult. The effects of DADLE were subdued by naltrindole (specific DOR antagonist) reaffirming the involvement of DOR in the process. Taken together these results promulgate the role of DADLE-induced DOR activation on improved hMSC survival, which signifies the plausible implications of DOR-activation in cell-transplantation therapies and tissue engineering aspect.

The delta opioid peptide D-Alanine 2, Leucine 5 Enkephaline (DADLE)-induces neuroprotection through cross-talk between the UPR and pro-survival MAPK-NGF-Bcl2 signaling pathways via modulation of several micro-RNAs in SH-SY5Y cells subjected to ER stress.

Parkinson's disease (PD) is the second most progressive neurodegenerative disease characterized by the loss of dopaminergic neurons and accumulation of misfolded proteins in endoplasmic reticulum (ER) leading to activation of the unfolded protein response (UPR). In the present study, we aimed to determine the potential survival effect of the delta opioid neuro-peptide D-Alanine 2, Leucine 5 Enkephaline (DADLE), and its mechanism in dopaminergic SH-SY5Y cells which were subjected to ER stress. In this cellular model of PD, enhanced cell survivability was observed on DADLE treatment (but not with µ and κ opioid agonists) along with concomitant down regulation of the UPR stress sensors and protein aggregates. The study found increased phosphorylation of MEK-1, which leads to activation of MAP kinase as well as enhanced expression of the pro-survival gene nerve growth factor and anti-apoptotic marker Bcl2. DADLE treatment could also significantly inhibit expression of the pro-apoptotic marker BIM. Next-generation sequence analysis revealed 93 micro (mi) RNAs to be differentially regulated following DADLE treatment in cells subjected to ER stress. Pathway prediction and previously published reports revealed that out of these 93 miRNAs, 34 can play a role in promoting cell survival. Specific modulation of two such miRNAs, namely miR-30c-2-3p and miR-200c, could partially reverse the positive survival effect induced by DADLE. Apart from the known miRNAs, various novel miRNAs were also observed following DADLE treatment which could also play a role in enhancing the survival of SH-SY5Y cells under ER stress.