[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.

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.

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 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.

[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.

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.

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.

Delta Opioid Receptor and Peptide: A Dynamic Therapy for Stroke and Other Neurological Disorders.

Research of the opioid system and its composite receptors and ligands has revealed its promise as a potential therapy for neurodegenerative diseases such as stroke and Parkinson's Disease. In particular, delta opioid receptors (DORs) have been elucidated as a therapeutically distinguished subset of opioid receptors and a compelling target for novel intervention techniques. Research is progressively shedding light on the underlying mechanism of DORs and has revealed two mechanisms of DOR neuroprotection; DORs function to maintain ionic homeostasis and also to trigger endogenous neuroprotective pathways. Delta opioid agonists such as (D-Ala2, D-Leu5) enkephalin (DADLE) have been shown to promote neuronal survival and decrease apoptosis, resulting in a substantial amount of research for its application as a neurological therapeutic. Most notably, DADLE has demonstrated significant potential to reduce cell death following ischemic events. Current research is working to reveal the complex mechanisms of DADLE's neuroprotective properties. Ultimately, our knowledge of the DOR receptors and agonists has made the opioid system a promising target for therapeutic intervention in many neurological disorders.

Delta opioid receptor agonist attenuates lipopolysaccharide-induced myocardial injury by regulating autophagy.

BACKGROUND: Previous studies have described the protective effects of DADLE on myocardial injury in sepsis. Recently, autophagy has been shown to be an innate defense mechanism in sepsis-related myocardial injury. However, whether DADLE has an pro-autophagic effect is yet to be elucidated. The present study aimed to investigate the effect of DADLE on the regulation of autophagy during sepsis. METHODS: Male mice were subjected to LPS or vehicle intraperitoneal injection. After LPS injection, mice received either DADLE, Naltrindole or vehicle. ELISA and JC-1 were used to evaluate the level cTnI and Mitochondrial membrane potential. Cardiac ultrastructural and autophagosomes were visualized by transmission electron microscopy. The relative protein levels were analyzed by Western blot. RESULTS: The results showed that treatment with DADLE both immediately or 4 h after LPS intraperitoneal injection could improve the survival rate of mice with endotoxemic. DADLE could ease myocardium ultrastructure injury induced by LPS, this cardioprotective effect was also seen in increased MMP levels, and decreased cTnI levels. Through observation of transmission electron microscopy and Western blot we have discovered that the amount of autophagosome and the expression of autophagy related protein LC3II, Beclin1 were significantly increased with DADLE treatment. DADLE promoted LPS-induced autophagosome maturation as indicated by the increased LAMP-1 protein level and decreased SQSTM1/p62 protein level. The selective δ-opioid receptor antagonist Naltrindole play an opposite effects. CONCLUSIONS: DADLE could improve the survival and protect myocardial dysfunction in mice with LPS-induced endotoxemia. This effect was related to the increase of autophagy.

The Delta-Opioid Receptor Agonist D-Ala2-D-Leu2-Enkephalin Enhances Hypoxic Depression of Excitatory Synaptic Transmission and Improves Recovery of Rat Cortical Neurons from Hypoxia in vitro.

We investigated the influence of the delta-opioid receptor-preferring agonist D-Ala2-D-Leu2-enkephalin (DADLE) in vitro during long- and short-term hypoxia on the single cortical neuron membrane currents, the postsynaptic currents (PSCs), and the postsynaptic potentials (PSPs) in rats. Rat cortical pyramidal neurons showed 2 distinct and prognostically relevant responses to hypoxia. Type A neurons that responded to hypoxia by an inward current, followed by a steady outward current, were shown to recover during subsequent reoxygenation. In contrast, type B neurons that responded by a steady inward current, indicative of gradual anoxic depolarisation, suffered irreversible membrane dysfunction and did not recover completely during reoxygenation. Pre-treatment with 1 µmol/l DADLE attenuated the hypoxic inward current and favored complete recovery of holding current and input resistance during reoxygenation, even when neurons were challenged by a second exposure to hypoxia. DADLE enhanced the inhibitory effect of hypoxia on PSPs and PSCs. We assume that this neuroprotective effect is transmitted by the additive effects of DADLE on the hypoxic PSP/PSC suppression, thereby inhibiting presynaptic glutamate release.

Correction of Negative Effect of Antenatal Hypoxia on Liver Tissue Homeostasis in Newborn Albino Rats with Opioid Peptides.

We studied the possibility of correction of the negative effects of antenatal hypoxia on the liver tissue homeostasis in 7-day-old albino rats by administration of opioid peptides in a dose of 100 µg/kg on postnatal days 2-6. Administration of mixed µ/δ-opioid receptor agonist Dalargin neutralized deviations of gravimetric indicators, parameters of proliferative activity, and activity of the nucleolar apparatus of hepatocytes. Administration of the non-opiate Leu-enkephalin analogue did not normalize gravimetric parameters and nucleolar apparatus parameters, however, it significantly increased the pool of proliferating hepatocytes. Both peptides significantly reduced the intensity of free radical oxidation, improved antioxidant antiradical defense and resistance to peroxidation in the liver tissue of animals subjected to antenatal hypoxia.

[ЕXPERIMENTAL ESTIMATION OF THE LOCAL APPLICATION EFFICACY OF BIOLOGICAL STIMULATOR FOR THE SOFT TISSUES REPARATION IN TREATMENT OF CHRONIC GASTRIC ULCERS].

Results of experimental investigation for studying of the local application possibility for the reparative processes stimulators, including autologic purified lipoaspirate, dalargin and a platelet­rich plasma (PRP) for treatment of chronic gastric ulcers, were analyzed. The advantages of PRP application, as the growth factors donator, independently and in combination with dalargin, were proved. The PRP capacity to stimulate the fibroblasts activity and creation of vessels in young connective tissue was proved, what have leaded to the ulcer tissues oxygenation improvement and cellular proliferation promotion, acceleration of the connective tissue maturation, and the ulcer healing.

Bradykinin controls pool size of sensory neurons expressing functional δ-opioid receptors.

Analgesics targeting the δ-opioid receptor (DOR) may lead to fewer side effects than conventional opioid drugs, which mainly act on µ-opioid receptors (MOR), because of the less abundant expression of DOR in the CNS compared with MOR. Analgesic potential of DOR agonists increases after inflammation, an effect that may be mediated by DOR expressed in the peripheral sensory fibers. However, the expression of functional DOR at the plasma membrane of sensory neurons is controversial. Here we have used patch-clamp recordings and total internal reflection fluorescence microscopy to study the functional expression of DOR in sensory neurons from rat trigeminal (TG) and dorsal root ganglia (DRG). Real-time total internal reflection fluorescence microscopy revealed that treatment of TG and DRG cultures with the inflammatory mediator bradykinin (BK) caused robust trafficking of heterologously expressed GFP-tagged DOR to the plasma membrane. By contrast, treatment of neurons with the DOR agonist [d-Ala(2), d-Leu(5)]-enkephalin (DADLE) caused a decrease in the membrane abundance of DOR, suggesting internalization of the receptor after agonist binding. Patch-clamp experiments revealed that DADLE inhibited voltage-gated Ca(2+) channels (VGCCs) in 23% of small-diameter TG neurons. Pretreatment with BK resulted in more than twice as many DADLE responsive neurons (54%) but did not affect the efficacy of VGCC inhibition by DADLE. Our data suggest that inflammatory mediator-induced membrane insertion of DOR into the plasma membrane of peripheral sensory neurons may underlie increased DOR analgesia in inflamed tissue. Furthermore, the majority of BK-responsive TG neurons may have a potential to become responsive to DOR ligands in inflammatory conditions.

Use of halogenated precursors to define a transcription time window after treatment with hypometabolizing molecules.

A new method is proposed which combines the high spatial resolution of transmission electron microscopy with information on the dynamics of transcription. Incorporation of two different RNA precursors was used to define a time transcription window on cultured cells treated with hypometabolizing peptides which are known to modulate transcription. This procedure allows detecting a single fibril of newly synthesized RNA in the time range in which it is transcribed.

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.

Induction of torpor: mimicking natural metabolic suppression for biomedical applications.

Mammalian hibernation consists of periods of depressed metabolism and reduced body temperature called "torpor" that are interspersed by normothermic arousal periods. Numerous cellular processes are halted during torpor, including transcription, translation, and ion homeostasis. Hibernators are able to survive long periods of low blood flow and body temperature followed by rewarming and reperfusion without overt signs of organ injury, which makes these animals excellent models for application of natural protective mechanisms to human medicine. This review examines efforts to induce torpor-like states in non-hibernating species using pharmacological compounds. Elucidating the underlying mechanisms of natural and pharmacologically induced torpor will speed the development of new clinical approaches to treat a variety of trauma and stress states in humans.

Role of connexin-43 in protective PI3K-Akt-GSK-3ß signaling in cardiomyocytes.

Sarcolemmal connexin-43 (Cx43) and mitochondrial Cx43 play distinct roles: formation of gap junctions and production of reactive oxygen species (ROS) for redox signaling. In this study, we examined the hypothesis that Cx43 contributes to activation of a major cytoprotective signal pathway, phosphoinositide 3-kinase (PI3K)-Akt-glycogen synthase kinase-3ß (GSK-3ß) signaling, in cardiomyocytes. A δ-opioid receptor agonist {[d-Ala(2),d-Leu(5)]enkephalin acetate (DADLE)}, endothelin-1 (ET-1), and insulin-like growth factor-1 (IGF-1) induced phosphorylation of Akt and GSK-3ß in H9c2 cardiomyocytes. Reduction of Cx43 protein to 20% of the normal level by Cx43 small interfering RNA abolished phosphorylation of Akt and GSK-3ß induced by DADLE or ET-1 but not that induced by IGF-1. DADLE and IGF-1 protected H9c2 cells from necrosis after treatment with H(2)O(2) or antimycin A. The protection by DADLE or ET-1, but not that by IGF-1, was lost by reduction of Cx43 protein expression. In contrast to Akt and GSK-3ß, PKC-ε, ERK and p38 mitogen-activated protein kinase were phosphorylated by ET-1 in Cx43-knocked-down cells. Like diazoxide, an activator of the mitochondrial ATP-sensitive K(+) channel, DADLE and ET-1 induced significant ROS production in mitochondria, although such an effect was not observed for IGF-1. Cx43 knockdown did not attenuate the mitochondrial ROS production by DADLE or ET-1. Cx43 was coimmunoprecipitated with the ß-subunit of G protein (Gß), and knockdown of Gß mimicked the effect of Cx43 knockdown on ET-1-induced phosphorylation of Akt and GSK-3ß. These results suggest that Cx43 contributes to activation of class I(B) PI3K in PI3K-Akt-GSK-3ß signaling possibly as a cofactor of Gß in cardiomyocytes.

Role of δ2 opioid receptor in cardioprotection against hypoxia-reoxygenation injury.

Several lines of in vivo evidence demonstrated that activation of δ-opioid receptors (ORs) with agonists mimics the cardioprotective effect of ischemic preconditioning. However, the subtypes of ORs involved and the molecular and cellular mechanisms are not entirely clear. To investigate the significance of the contribution by δ ORs to cardiomyocyte survival, we used an in vitro model of hypoxia/reoxygenation (H/R) in primary cultures of neonatal rat cardiomyocytes to study the role of different δ ORs in cardiomyocyte apoptosis and the relevant downstream signaling pathway. The results showed that apoptosis in neonatal cardiomyocytes induced by H/R was reversed by δ2 OR agonist, deltorphin E but not by δ1 OR agonist DPDPE; the deltorphin E-induced cytoprotection was totally abrogated by the MEK inhibitor PD98059 and overexpression of dominant interfering form of MEK1; in contrast, overexpression of constitutive active form of MEK1 exerted a similar protective effect as deltorphin E. These results suggest that δ2 OR, but not δ1 OR, plays a key role in preventing cardiomyocytes from apoptosis during H/R injury, which is mainly mediated by the MEK/ERK1/2 pathway.

[DADLE suppresses the proliferation of human liver cancer HepG2 cells by activation of PKC pathway and elevates the sensitivity to cis-diammine dichloridoplatium].

OBJECTIVE: To investigate the effect of DADLE, a δ-opioid receptor agonist, on the proliferation of human liver cancer HepG2 cells and explore the mechanism involving PKC pathway. METHODS: HepG2 cells were treated with DADLE at different doses (0.01, 0.1, 1.0 and 10 µmol/L). Cell viability was determined using methyl thiazolyl terazolium (MTT) assay. The expression of PKC mRNA and p-PKC protein were examined by RT-PCR and Western blot assay. After treated separately with DADLE plusing NAL or PMA, the cell cycle of HepG2 cells was analyzed by flow cytometer. MTT was used to detect their proliferation capacity and Western blot was used to examine the p-PKC expression. The growth inhibitory rate of HepG2 cells treated with DADLE and cis-diammine dichloridoplatinum (CDDP) was analyzed. RESULTS: DADLE at different concentrations showed an inhibitory effect on the proliferation of HepG2 cells though inhibiting the expression of PKC mRNA and p-PKC protein. The results of flow cytometry showed that compared with the control group, the percentage of S + G(2)/M cells in DADLE-treated group was lowered by 3.94% (P < 0.01). Meanwhile, after treated with NAL and PMA, the percentage was elevated by 3.22% and 3.63%, respectively (P < 0.01). The MTT and Western blot assays showed that compared with the control group, the values of A570 and p-PKC protein levels in the HepG2 cells of DADLE-treated group were significantly decreased (P < 0.01). After treatment with NAL and PMA, the values of A570 and p-PKC protein levels were elevated significantly (P < 0.01). The growth inhibitory rate of DADLE + CDDP group was 79.9%, significantly lower than 25.2% and 43.2% of the DADLE and CDDP groups, respectively. CONCLUSIONS: Activation of δ-opioid receptor by DADLE inhibits the apoptosis of human liver cancer HepG2 cells. The underlying mechanism may be correlated with PKC pathway. DADLE can enhance the chemosensitivity of HepG2 cells to CDDP.

Effects of peripheral µ, δ, and Κ-opioid receptor agonists on the levels of anxiety and motor activity of rats.

The effects of intragastric administration of µ-, δ, and Κ-opioid receptor agonists DAMGO, DADLE, and ICI 204,448, respectively, on the anxiety and motor activity of rats in an elevated plus-maze were studied. Peripheral administration of ICI 204,448 produced an anxiolytic effect, but had no effect on motor activity of rats. DAMGO and DADLE reduced motor activity; DADLE also increased anxiety. The data on the opposite effects of ICI 204,448 and DADLE on anxiety confirmed our previous hypothesis on the interactions between the central and peripheral components of the endogenous opioid system.