enkephalin--leucine-2-alanine and Hypoxia

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, **

Topics: Analgesics, Opioid; Animals; Animals, Newborn; Apoptosis; Caspase 3; Enkephalin, Leucine-2-Alanine; Humans; Hypoxia; Ischemic Preconditioning, Myocardial; Models, Biological; Myocardial Contraction; Myocardial Reperfusion Injury; Myocytes, Cardiac; Rats; Receptors, Opioid, delta; Species Specificity; Tissue Engineering; Troponin I

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.

Topics: Animals; Animals, Newborn; Antioxidants; Body Weight; Cell Nucleus; Enkephalin, Leucine-2-Alanine; Female; Free Radicals; Hepatocytes; Homeostasis; Hypoxia; Liver; Opioid Peptides; Organ Size; Oxidative Stress; Rats; Rats, Wistar

[d-Ala2, d-Leu5] enkephalin (DADLE) is a synthetic δ-opioid agonist that induces hibernation and promotes survival of neurons and glial cells in the central nervous system. Several mechanisms for the attenuation of hypoxic injury have been suggested, including control of intracellular signaling pathways via the δ-opioid receptor (DOR). However, the cellular and molecular mechanisms of DADLE in hypoxic injury are largely unknown. To investigate neuronal injury after oxygen-deprivation (OD) and DOR stimulation by DADLE, we used a lactate dehydrogenase assay, MTT assay, and immunofluorescence live/dead staining. And we used cDNA microarrays to investigate the influence of DADLE exposure on transcription after OD in rat cortical glial and neuronal co-culture. DADLE reduced neuronal injury after 24 h of OD. Preconditioning with DADLE before 24 h hypoxia exposure also altered gene expression in comparison with 24h OD without pretreatment. After DADLE exposure and hypoxia, 1917 of 39,511 genes (4.9%) were significantly induced or repressed at least 2.5-fold. Assigning differentially expressed ESTs (expressed sequence tags) to molecular functional groups revealed that DADLE affected many pathways including apoptosis, intracellular ion homeostasis, molecular chaperones, and glucose metabolism. We observed a coordinated change in expression of many genes (increased expression of potentially protective genes and decreased expression of potentially harmful genes) after DADLE exposure. A comprehensive list of regulated genes should prove valuable in advancing our understanding of the neuroprotective mechanisms of DADLE under OD.

Topics: Animals; Animals, Newborn; Cell Hypoxia; Cerebral Cortex; Coculture Techniques; Enkephalin, Leucine-2-Alanine; Genomics; Hypoxia; Ischemic Preconditioning; Neuroglia; Neurons; Oligonucleotide Array Sequence Analysis; Rats; Rats, Sprague-Dawley; Receptors, Opioid, delta

Past work has shown that delta-opioid receptor (DOR) activation by [D-Ala(2),D-Leu(5)]-enkephalin (DADLE) attenuated the disruption of K(+) homeostasis induced by hypoxia or oxygen-glucose deprivation (OGD) in the cortex, while naltrindole, a DOR antagonist blocked this effect, suggesting that DOR activity stabilizes K(+) homeostasis in the cortex during hypoxic/ischemic stress. However, several important issues remain unclear regarding this new observation, especially the difference between DOR and other opioid receptors in the stabilization of K(+) homeostasis and the underlying mechanism. In this study, we asked whether DOR is different from micro-opioid receptors (MOR) in stabilizing K(+) homeostasis and which membrane channel(s) is critically involved in the DOR effect. The main findings are that (1) similar to DADLE (10 microM), H-Dmt-Tic-NH-CH (CH(2)--COOH)-Bid (1-10 microM), a more specific and potent DOR agonist significantly attenuated anoxic K(+) derangement in cortical slice; (2) [D-Ala(2), N-Me-Phe(4), glycinol(5)]-enkephalin (DAGO; 10 microM), a MOR agonist, did not produce any appreciable change in anoxic disruption of K(+) homeostasis; (3) absence of Ca(2+) greatly attenuated anoxic K(+) derangement; (4) inhibition of Ca(2+)-activated K(+) (BK) channels with paxilline (10 microM) reduced anoxic K(+) derangement; (5) DADLE (10 microM) could not further reduce anoxic K(+) derangement in the Ca(2+)-free perfused slices or in the presence of paxilline; and (6) glybenclamide (20 microM), a K(ATP) channel blocker, decreased anoxia-induced K(+) derangement, but DADLE (10 microM) could further attenuate anoxic K(+) derangement in the glybenclamide-perfused slices. These data suggest that DOR, but not MOR, activation is protective against anoxic K(+) derangement in the cortex, at least partially via an inhibition of hypoxia-induced increase in Ca(2+) entry-BK channel activity.

Topics: Animals; Calcium; Cerebral Cortex; Enkephalin, Leucine-2-Alanine; Homeostasis; Hypoxia; Large-Conductance Calcium-Activated Potassium Channels; Male; Mice; Mice, Inbred C57BL; Neurons; Peptides; Potassium; Receptors, Opioid, delta; Receptors, Opioid, mu

Hypoxia preconditioning (HPC), rapid or delayed, has been reported to induce neuroprotection against subsequent severe stress. Because delta-opioid receptor (DOR) plays an important role in delayed HPC-induced neuroprotection against severe hypoxic injury, we asked whether DOR is also involved in the rapid HPC-induced neuroprotection.. Cultured rat cortical neurons at culture days 8 to 9 were exposed to a short-term hypoxia (1% O2 for 30 minutes) to induce HPC followed by 30-minute normoxia before exposing to glutamate toxicity (100 micromol/L; 4 hours). Neuronal viability was assessed by lactate dehydrogenase leakage and morphological assessment. Protein and mRNA levels of DOR were detected by receptor binding and RT-PCR, respectively. Naltrindole was used to block DOR. Developmental changes in NMDA receptor expression was measured by Western blots.. HPC significantly reduced the glutamate-induced neuronal injury. Receptor binding showed that HPC increased DADLE (a DOR ligand) binding density in the cultured cortical neurons by >90% over control level (P<0.05), although RT-PCR did not detect any appreciable change in DOR mRNA. DOR inhibition with naltrindole had no effect on neuronal injury and completely abolished the HPC-induced neuroprotection. In contrast to HPC-induced increase in DADLE binding density, prolonged hypoxia caused severe neuronal injury with a significant decrease in DADLE binding density and DOR mRNA level.. DOR is involved in neuroprotection induced by rapid HPC in cortical neurons.

Topics: Animals; Cells, Cultured; Cellular Senescence; Cerebral Cortex; Enkephalin, Leucine-2-Alanine; Glutamic Acid; Hypoxia; Ischemic Preconditioning; L-Lactate Dehydrogenase; N-Methylaspartate; Naltrexone; Narcotic Antagonists; Neurons; Rats; Receptors, Opioid, delta; RNA, Messenger; Time Factors; Up-Regulation

Our previous work shows that delta-opioid receptor (DOR) protects cortical neurons from hypoxic insults. Since an enhanced anaerobic capacity is important for neurons to adapt to the reduction of oxidative phosphorylation, we asked whether DOR plays a role in neuronal regulation of anaerobic capacity, thus protecting neurons from O(2) deprivation. Indeed, there is evidence suggesting that DOR may regulate protein kinase A (PKA) and C (PKC), which are involved in regulation of lactate dehydrogenase (LDH). However, little is known regarding the role of DOR and protein kinases in the regulation of glycolytic and related enzymes. As a first step, the present studies were performed in primary cultures of rat cortical neurons to clarify two issues: (1) Are protein kinases involved in the regulation of LDH activity in hypoxia? and (2) Does DOR affect LDH activity in hypoxic neurons? The results showed that PKC activation yielded substantial increases in normoxic LDH activity and significantly augmented LDH activity in hypoxic neurons, while PKC inhibition decreased LDH activity in both normoxic and hypoxic neurons. PKA activation significantly increased LDH activity in normoxic neurons and further elevated LDH activity in hypoxic neurons. However, PKA inhibition did not decrease in LDH activity in either normoxic or hypoxic neurons. Although DOR inhibition slightly reduced LDH activity in normoxia, DOR activation or inactivation did not alter LDH activity in hypoxic neurons. These data suggest that in cortical neurons, (i) PKC up-regulates LDH activity and plays an important role in its up-regulation during hypoxia; (ii) PKA is less likely involved in the regulation of LDH activity during hypoxia although its stimulation may slightly increase LDH activity and (iii) DOR does not contribute to LDH activity up-regulation during hypoxia.

Topics: Animals; Cell Hypoxia; Cells, Cultured; Cerebral Cortex; Cyclic AMP; Embryo, Mammalian; Enkephalin, Leucine-2-Alanine; Enzyme Activators; Enzyme Inhibitors; Female; Hypoxia; L-Lactate Dehydrogenase; Naltrexone; Neurons; Pregnancy; Protein Kinases; Rats; Rats, Sprague-Dawley; Receptors, Opioid, delta; Time Factors

Mammalian hibernation is mediated by humoral agonists of the delta opioid receptor (DOR). Moreover, transfer of either humoral or synthetic DOR agonists to non-hibernators reportedly induces a state of improved myocardial ischemic tolerance.. To determine whether the DOR agonist D-Ala 2, D-Leu 5, enkephalin (DADLE) similarly elicits protection in noncardiac-i.e., mesenteric-tissue.. In Protocols 1 and 2, the authors developed and characterized an in vitro model of mesenteric ischemia/reperfusion in isolated rabbit jejunum by documenting the effect of increasing ischemic duration (0 to 120 minutes) and the relative importance of glucose and/or oxygen deprivation on the evolution of jejunal injury. In Protocol 3, jejunal segments were randomized to receive either no treatment (controls) or 15 minutes of pretreatment with 1 microM DADLE, followed by 60 minutes of simulated ischemia and 30 minutes of reperfusion. Jejunal injury was quantified by repeated, time-matched assessment of peak contractile force evoked by 1 microM acetylcholine (all protocols) and delineation of tissue necrosis (Protocol 1).. Development of significant jejunal injury required combined oxygen/glucose deprivation. Moreover, there was a direct relationship between ischemic duration and tissue injury, and a significant inverse correlation between reperfusion contractile force (% of baseline) and the extent of smooth muscle necrosis (r(2) = 0.87; p < 0.01). Most notably, mesenteric ischemia/reperfusion injury was attenuated by DADLE: reperfusion contractile force was 47 +/- 5% versus 36 +/- 5% in DADLE-treated versus control segments (p < 0.01).. Treatment with the delta opioid agonist DADLE increases ischemic tolerance of isolated rabbit jejunum.

Topics: Animals; Disease Models, Animal; Dose-Response Relationship, Drug; Enkephalin, Leucine-2-Alanine; Glucose; Hypoxia; In Vitro Techniques; Ischemia; Jejunum; Muscle Contraction; Muscle, Smooth; Rabbits; Receptors, Opioid, delta; Reperfusion Injury; Splanchnic Circulation

Using radiography with H-thymidine method we studied the synthesis of DNA process in pyloric parts of stomach epithelium in white rats, which have been five-fold effected by different kinds of stressors against a background of dalargin injections. In the first hour after animals were stressed, DNA synthesis was depressed. Dalargin injections caused DNA synthesis normalization in the first hour after hypoxia and hyperthermia. Since 24 hours after hyperthermia and immobilization against a background of dalargin injections the normalization of DNA synthesis took place, and after hypoxia the post-stressing IMN activation was growing week. One of the mechanisms of dalargin correction of DNA synthesis breach under the influence of stressors is a stabilization of noradrenaline and histamine concentration in tissue of the stomach.

Topics: Animals; Cell Division; DNA; Enkephalin, Leucine-2-Alanine; Epithelial Cells; Epithelium; Fever; Histamine; Hypoxia; Immobilization; Male; Norepinephrine; Rats; Stomach; Stress, Physiological

Development of posthypoxic and reoxygenation depression of cardiac activity following a time-portioned disconnection of the apparatus for artificial ventilation of the lungs in rats was restricted by a preventive intravenous infusion of a synthetic opioid peptide--dalargin. Resistance to a stressor effect of hypoxia (ischemia-reperfusion) which was assessed by the degree of restoration of the integral index of the blood circulation--cardiac output could be mediated by correction of Ca-homeostasis of cardiomyocytes by means of dalargin. Elimination of all side effects with a blocker of opioid receptors--naloxon points to a possibility of realization of a protective antihypoxic action.

Topics: Acute Disease; Animals; Asphyxia; Disease Models, Animal; Drug Evaluation, Preclinical; Enkephalin, Leucine-2-Alanine; Hemodynamics; Hypoxia; Myocardial Reperfusion Injury; Rats; Rats, Inbred Strains; Respiration, Artificial; Time Factors

Topics: Adrenal Glands; Animals; beta-Endorphin; Brain; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, Leucine; Enkephalin, Leucine-2-Alanine; Enkephalin, Methionine; Enkephalins; Hypoxia; Lactates; Male; Mice; Rats; Rats, Inbred Strains

The effects of selective opioid receptor agonists and antagonists on neural discharge recorded from carotid body arterial chemoreceptors in vivo were studied in anaesthetized cats. Mean ID50 values were determined for each agonist and used to assess chemodepressant potency on intracarotid (i.c.) injection in animals artificially ventilated with air. [Met]enkephalin, [Leu]enkephalin, [D-Ala2, D-Leu5]enkephalin and [D-Pen2, D-Pen5]enkephalin were more potent chemodepressants than [D-Ala2, Me-Phe4, Gly-ol5]enkephalin, dynorphin (1-8) or ethylketocyclazocine; morphiceptin (mu-agonist) was inactive. The rank order of potency was compatible with the involvement of delta-opioid receptors in opioid-induced depression of chemosensory discharge. ICI 154129, a delta-opioid receptor antagonist, was used in fairly high doses and caused reversible dose-related antagonism of chemodepression induced by [Met]enkephalin. It also antagonized depression caused by single doses of [Leu]enkephalin, [D-Ala2, D-Leu5]enkephalin, [D-Ala2, Me-Phe4, Gly-ol5]enkephalin or dynorphin (1-8). ICI 174864, a more potent and selective delta-opioid receptor antagonist, also antagonized chemodepression induced by [Met]enkephalin or by the selective delta-receptor agonist [D-Pen2, D-Pen5]enkephalin. Comparison of background or 'spontaneous' chemosensory discharge during the 30 min periods immediately before and after injecting ICI 174864 (0.1-0.2 mg kg-1 i.c.) showed a significant increase in discharge in one experiment, but in four others discharge was either unaffected or decreased after the antagonist, which argues against a toxic depression of chemosensors by endogenous opioids under resting conditions in our preparation. Sensitivity of the carotid chemoreceptors to hypoxia (ventilating with 10% O2) was increased significantly after ICI 174864, which could be taken as evidence that endogenous opioids depress chemosensitivity during hypoxia. In contrast, responsiveness to hypercapnia was reduced after the antagonist, implying that endogenous opioids may potentiate chemoreceptor sensitivity during hypercapnia. The results obtained using 'selective' agonists and antagonists provide evidence that depression of chemosensory discharge caused by injected opioids involves a delta type of opioid receptor within the cat carotid body. Endogenous opioids may modulate arterial chemoreceptor sensitivity to physiological stimuli such as hypoxia and hypercapnia.

Topics: Animals; Carotid Body; Cats; Chemoreceptor Cells; Cyclazocine; Dose-Response Relationship, Drug; Dynorphins; Endorphins; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, D-Penicillamine (2,5)-; Enkephalin, Leucine; Enkephalin, Leucine-2-Alanine; Enkephalin, Methionine; Enkephalins; Ethylketocyclazocine; Hypercapnia; Hypoxia; Peptide Fragments; Receptors, Opioid