This study was undertaken to investigate the effects of [D-Ala2, D-Leu5]-enkephalin (DADLE) on the spontaneous activity of medial vestibular nuclear neurons of the rat. Sprague-Dawley rats, aged 14 to 16 days, were anesthetized with ether and decapitated. After enzymatic digestion, the brain stem portion of medial vestibular nuclear neuron was obtained by micropunching. The dissociated neurons were transferred to a recording chamber mounted on an inverted microscope, and spontaneous action potentials were recorded by standard patch-clamp techniques. The spontaneous action potentials were increased by DADLE in 12 cells and decreased in 3 cells. The spike frequency and resting membrane potential of these cells were increased by DADLE. The depth of afterhyperpolarization was not affected by DADLE. The potassium currents were decreased in 20 cells and increased in 5 cells. These results suggest that DADLE increases the neuronal activity of the medial vestibular nuclear neurons by altering resting membrane potential.
Action Potentials ; Animals ; Brain Stem* ; Digestion ; Enkephalin, Leucine-2-Alanine ; Ether ; Membrane Potentials ; Neurons* ; Patch-Clamp Techniques ; Potassium ; Rats* ; Rats, Sprague-Dawley

PURPOSE: Oxygen is indispensable for survival and aerobic metabolism in all mammalian cells. Inadequate oxygen triggers a multifaceted cellular response negatively impacting important physiological functions which are observed in clinical diseases such as stroke, drowning, cardiac arrest, hazardous gas poisoning, myocardial infarction and vascular dementia. In this study, we investigated the neuroprotective effect of a synthetic delta-opioid agonist, [D-Ala2, D-Leu5] enkephalin (DADLE), and its role in ischemic neuronal injury. METHODS: This experiment was conducted in vitro using a primary culture of rat cortical neurons. Ischemia induction was performed using a hypoxic chamber. To test the degree of neuronal viability, as protected by delta-opioid stimulation with DADLE under ischemia, we used three independent approaches including a lactate dehydrogenase assay, MTT assay, and an immunofluorescent staining assay for viable cells. In addition, the gene expressions of caspase-3 and heat shock protein 70 were analyzed using real-time PCR. RESULTS: Incubation of the cortical neurons with DADLE protected them from ischemia-induced cytotoxicity, as observed by all three independent viability assays. Also, we found that its neuroprotective effect might be related with suppression of the caspase-3 gene. CONCLUSION: The results of this study suggested that DADLE exhibits a neuroprotective effect against ischemia-induced neuronal cell death.
Animals ; Caspase 3 ; Cell Death ; Dementia, Vascular ; Drowning ; Enkephalin, Leucine-2-Alanine ; Enkephalins ; Gas Poisoning ; Gene Expression ; Heart Arrest ; HSP70 Heat-Shock Proteins ; Ischemia ; L-Lactate Dehydrogenase ; Myocardial Infarction ; Neurons ; Neuroprotective Agents ; Oxygen ; Rats ; Stroke

PURPOSE: Oxygen is indispensable for survival and aerobic metabolism in all mammalian cells. Inadequate oxygen triggers a multifaceted cellular response negatively impacting important physiological functions which are observed in clinical diseases such as stroke, drowning, cardiac arrest, hazardous gas poisoning, myocardial infarction and vascular dementia. In this study, we investigated the neuroprotective effect of a synthetic delta-opioid agonist, [D-Ala2, D-Leu5] enkephalin (DADLE), and its role in ischemic neuronal injury. METHODS: This experiment was conducted in vitro using a primary culture of rat cortical neurons. Ischemia induction was performed using a hypoxic chamber. To test the degree of neuronal viability, as protected by delta-opioid stimulation with DADLE under ischemia, we used three independent approaches including a lactate dehydrogenase assay, MTT assay, and an immunofluorescent staining assay for viable cells. In addition, the gene expressions of caspase-3 and heat shock protein 70 were analyzed using real-time PCR. RESULTS: Incubation of the cortical neurons with DADLE protected them from ischemia-induced cytotoxicity, as observed by all three independent viability assays. Also, we found that its neuroprotective effect might be related with suppression of the caspase-3 gene. CONCLUSION: The results of this study suggested that DADLE exhibits a neuroprotective effect against ischemia-induced neuronal cell death.
Animals ; Caspase 3 ; Cell Death ; Dementia, Vascular ; Drowning ; Enkephalin, Leucine-2-Alanine ; Enkephalins ; Gas Poisoning ; Gene Expression ; Heart Arrest ; HSP70 Heat-Shock Proteins ; Ischemia ; L-Lactate Dehydrogenase ; Myocardial Infarction ; Neurons ; Neuroprotective Agents ; Oxygen ; Rats ; Stroke

AIMTo study upon to serum deprivation if delta-opioid receptor activation has direct effect on cultured impaired cardiomyocytes survival.

METHODSMyocardial cells of neonatal rats were cultured in vitro. The cell viability was determined with crystal violet staining uptake. The percentage of S + G2 + M in cell cycle was determined by flow cytometry. Apoptosis rates were determined by flow cytometry (FCM). The expression of Caspase-3 were investigated by Western blotting.

RESULTSMyocardial cells of neonatal rats were cultured of serum-free in vitro, apoptotic index was significantly increased, the expression of Caspase-3 was significantly increased, free-serum induced apoptosis in cardiac myocytes after 48 h. At concentrations of 10 nmol x L(-1) - 10 micromol x L(-1), a delta opoid receptor agonist [D-Ala2, D-Leu5]-enkephalin DADLE promoted the myocardial cells survival, in a concentration-dependent manner. The optimal response was achieved at 0.1 micromol x L(-1), which increase survival index of cardiac myocyte, percentage of S + G2 + M in cell cycle, decrease apoptotic index of cardiac myocyte, and the expression activate caspase-3. Delta-opioid receptor antagonist naltrindole at 10 micromol x L(-1) inhibited the promoting effects of DADLE, which decrease survival index of cardiac myocyte, and percentage of S + G2 + M in cell cycle, increase apoptotic index of cardiac myocyte and the expression of Caspase-3.

CONCLUSIONThe protective of delta-opioid receptor activation can promote survival in cultured impaired myocardial cells.

Animals ; Animals, Newborn ; Apoptosis ; drug effects ; Caspase 3 ; metabolism ; Cell Survival ; drug effects ; Cells, Cultured ; Culture Media, Serum-Free ; Enkephalin, Leucine-2-Alanine ; pharmacology ; Female ; Male ; Myocytes, Cardiac ; cytology ; Rats ; Rats, Sprague-Dawley ; Receptors, Opioid, delta ; agonists

OBJECTIVETo 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.

METHODSHepG2 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.

RESULTSDADLE 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.

CONCLUSIONSActivation 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.

Antineoplastic Agents ; pharmacology ; Cell Cycle ; drug effects ; Cell Proliferation ; drug effects ; Cisplatin ; pharmacology ; Dose-Response Relationship, Drug ; Drug Resistance, Neoplasm ; Enkephalin, Leucine-2-Alanine ; administration & dosage ; pharmacology ; Hep G2 Cells ; Humans ; Naltrexone ; analogs & derivatives ; pharmacology ; Phosphorylation ; Protein Kinase C ; genetics ; metabolism ; RNA, Messenger ; metabolism ; Receptors, Opioid, delta ; agonists ; Signal Transduction ; Tetradecanoylphorbol Acetate ; analogs & derivatives ; pharmacology