Effect Of D-Ala2,D-Leu5]enkephalin Research Articles

D-Ala2, D-Leu5] Enkephalin Attenuates Hepatic Ischemia-Reperfusion Injury in Cirrhotic Rats.

Background and AimsHepatic ischemia–reperfusion injury (IRI) is a common phenomenon that occurs after liver transplantation and liver tumor surgery. It can cause liver dysfunction and recovery failure after liver surgery, even leading to acute liver failure. Our aim is to investigate the protective effect and related potential mechanism of [D-Ala2, D-Leu5] enkephalin (DADLE) treatment on hepatic IRI in cirrhotic livers of rats.MethodsThe models of liver cirrhosis and hepatic IRI were established with male Sprague–Dawley rats. DADLE at a dose series of 0.5, 1, or 5 mg·kg−1 was injected intravenously to rats 10 min prior hepatic ischemia, followed by a 6- h reperfusion. The serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), histological changes, and liver cell apoptosis were used to assess liver IRI. The optimal dose of DADLE was assessed by using the Suzuki score and ALT and AST levels. We repeated the hepatic IRI procedure on the optimal dose of the DADLE group and the delta opioid receptor (DOR) antagonist natrindole hydrochloride (NTD) injection group. Serum ALT and AST levels, histological staining, hepatic apoptosis, and serum levels of tumor necrosis factor alpha (TNF-α) and interleukin 1 β (IL-1β) were measured. The expression of protein kinase B (Akt) and its downstream proteins were evaluated by using quantitative real-time polymerase chain action (qRT-PCR) and Western blotting.ResultsCompared with the control group, DADLE treatment at a dose of 5 mg·kg−1 reduced the Suzuki score (mean: 5.8, range: 5.0–6.6 vs. mean: 8.0, range: 7.0–8.9), the ALT level (134.3 ± 44.7 vs. 247.8 ± 104.6), and the AST (297.1 ± 112.7 vs. 660.8 ± 104.3) level. DOR antagonist NTD aggravated hepatic IRI. Compared with the control group, DADLE treatment decreased the number of apoptosis cells and microphages and neutrophils, increased the expression of Akt and its mRNA to much higher levels, and upregulated the mRNA and protein expression of Bcl-2 and Bcl-2-associated death promoter (BAD).ConclusionDADLE treatment at a dose of 5 mg·kg−1 injected intravenously 10 min prior hepatic ischemia could contain rats’ hepatic IRI by activating DOR in cirrhotic livers. The effects of DADLE could be offset by NTD. The potential molecular mechanism seems to be involved in the phosphatidylinositol-3-kinase (PI3K)/Akt pathway.

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

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

Opioid inhibition of rat medial vestibular nucleus neurones in vitro and its dependence on age

Extracellular and whole-cell patch clamp intracellular recordings were made from rat medial vestibular nucleus (MVN) neurones in vitro, and their responses to selective mu-, kappa- and delta-opioid receptor agonists and antagonists were examined. Of 127 neurones tested, the large majority were inhibited in a dose-dependent manner by the delta-opioid receptor agonists [D-Ala2, D-Leu5]-enkephalin (DADLE) and [D-Pen2, Pen5]-enkephalin (DPLPE). The mu-opioid receptor agonist morphine and the kappa-receptor agonist U50,488 did not affect the tonic discharge rate of any of the 63 MVN cells tested. The delta-receptor antagonist naltrindole effectively antagonised the inhibitory effects of DADLE and DPLPE. Weak excitatory responses to high doses of DADLE were seen in only two MVN cells. These results demonstrate the presence of delta- but not mu- or kappa-opioid receptors on tonically active MVN neurones. Whole-cell intracellular recordings from MVN cells in a current clamp showed that the DADLE-induced inhibition was accompanied by membrane hyperpolarisation and decrease in input resistance, while voltage clamp experiments showed that DADLE induced an outward membrane current that was reduced but not abolished by 20 mM tetraethylammonium bromide. Thus the mechanisms of action of DADLE in inhibiting MVN cells involve the potentiation of outward K currents, in a similar way to the effects of opioids in other areas of brain. The inhibitory effects of DADLE increased linearly with age, so that the responses to DADLE in the youngest animals used here (60-80 g, approx. 3 weeks of age) were relatively small, increasing significantly over the following 2-3 weeks. This age-dependence may be due to post-natal changes in the density of delta-opiate receptors or the efficacy of the signalling pathways activated by them in the MVN cells over this time.

Dual signal transduction through delta opioid receptors in a transfected human T-cell line.

Opiates are known to function as immunomodulators, in part by effects on T cells. However, the signal transduction pathways mediating the effects of opiates on T cells are largely undefined. To determine whether pathways that regulate free intracellular calcium ([Ca2+]i) and/or cAMP are affected by opiates acting through delta-type opioid receptors (DORs), a cDNA encoding the neuronal DOR was expressed in a stably transfected Jurkat T-cell line. The DOR agonists, deltorphin and [D-Ala2, D-Leu5]-enkephalin (DADLE), elevated [Ca2+]i, measured by flow cytofluorometry using the calcium-sensitive dye, Fluo-3. At concentrations from 10(-11)-10(-7) M, both agonists increased [Ca2+]i from 60 nM to peak concentrations of 400 nM in a dose-dependent manner within 30 sec (ED50 of approximately 5 x 10(-9) M). Naltrindole, a selective DOR antagonist, abolished the increase in [Ca2+]i, and pretreatment with pertussis toxin was also effective. To assess the role of extracellular calcium, cells were pretreated with EGTA, which reduced the initial deltorphin-induced elevation of [Ca2+]i by more than 50% and eliminated the second phase of calcium mobilization. Additionally, the effect of DADLE on forskolin-stimulated cAMP production was determined. DADLE reduced cAMP production by 70% (IC50 of approximately equal to 10(-11) M), and pertussis toxin inhibited the action of DADLE. Thus, the DOR expressed by a transfected Jurkat T-cell line is positively coupled to pathways leading to calcium mobilization and negatively coupled to adenylate cyclase. These studies identify two pertussis toxin-sensitive, G protein-mediated signaling pathways through which DOR agonists regulate the levels of intracellular messengers that modulate T-cell activation.

Ethanol-induced increase in endogenous dopamine release may involve endogenous opiates.

The effect of opiate peptides on basal and potassium-stimulated endogenous dopamine (DA) release from striatal slices was studied in vitro. Dual stimulation of the striatal slices gave a reproducible increase in DA release that was calcium dependent. Addition of the delta-opiate receptor agonists Met5-enkephalin, [D-Ala2,D-Leu5]enkephalin (DADLE), and [D-Ser2]Leu-enkephalin-Thr (DSLET), increased the basal DA release without affecting potassium-stimulated release in a dose-dependent manner. The effect of DADLE was antagonized by the addition of naloxone. In contrast, the mu-opioid receptor agonist [D-Ala2,N-MePhe4,Gly-ol5]enkephalin (DAGO) and the epsilon-opioid agonist beta-endorphin inhibited the stimulated DA release without changing the basal release. The inhibitory effect of DAGO on potassium-stimulated release was antagonized by naloxone. The addition of ethanol (75 mM) to the incubation media produced a delayed increase of both the basal and stimulated DA release. There was no change in stimulated DA release when the change in basal release was subtracted, suggesting that ethanol produced a dose-dependent, selective increase in basal DA release. Naloxone and the selective delta-opiate antagonist ICI 174864 inhibited the ethanol-induced increase in basal DA release. Naloxone and ICI 174864 added alone did not alter either basal or stimulated DA release. We therefore suggest that the ethanol-induced increase in basal DA release is an indirect effect involving an endogenous delta-opiate agonist.

Inhibitory modulation of fast and slow Ca 2+-currents in neuroblastoma × glioma cells during differentiation

Mouse neuroblastoma × rat glioma hybrid cells (N × G, 108CC15) were used to study the inhibitory effects of the synthetic opioid d-Ala 2- d-Leu 5-enkephalin (DADLE), somatostatin, adrenaline- α 2 and angiotensin II on voltage-dependent Ca 2+-currents (I Ca) using the patch-clamp technique in the whole-cell configuration mode. The inhibitory effects could be abolished by pretreatment of N × G cells with pertussis toxin or intracellular infusion of GDP βS indicating an involvement of a pertussis toxin sensitive GTP-binding protein (G-protein), presumably G o. The effect of DADLE, the strongest inhibitor of I Ca, was studied during dibutyryl cyclic AMP (dBcAMP) induced differentiation. Using ω-conotoxin GVIA (ω-CTX) and methoxyverapamil (D600) as specific Ca 2+-channel blockers of the N- and L-type Ca 2+-channels, it was found that in N × G cells DADLE predominantly induces inhibition of T- and N-type Ca 2+-channels.

Opioid receptor desensitization in NG 108-15 cells: Differential effects of a full and a partial agonist on the opioid-dependent GTPase

Opioid-receptor binding and the opioid-mediated stimulation of low K m GTPase and inhibition of adenylate cyclase were studied in membranes derived from NG 108-15 cells pretreated with either the opioid peptide [ d-Ala 2, d-Leu 5]enkephalin (DADLE) or morphine. Pretreatment with DADLE resulted in a concentration-dependent loss of responsiveness of GTPase to the peptide; this effect was entirely accounted for by a reduction in the maximal stimulation produced acutely by DADLE, without changes in the ec 50, of the peptide, indicating a non-competitive type of desensitization. The degree of desensitization of GTPase was similar after one and 24 hr of pretreatment with DADLE, indicating that the process occurs rapidly. In contrast, morphine, which was 70–80% as potent as DADLE in stimulating GTPase and inhibiting adenylate cyclase in acute conditions, induced only a minimal desensitization of the opioid-GTPase system and, in contrast to DADLE, did not desensitize adenylate cyclase. Pretreatment with DADLE for one hour led to a decrease in opioid receptor density which was quantitatively similar to the degree of desensitization of GTPase: both these effects of DADLE were antagonized to a similar extent when morphine was also present in the pretreatment. Thus, desensitization of the opioid-stimulated GTPase appears to be correlated with down-regulation of the opioid receptor. Moreover, these findings suggest that partial agonists cannot induce this process.

Possible mechanisms of enkephalin action on hippocampal CA1 pyramidal neurons

(1) Intracellular and extracellular recordings were made from CA1 pyramidal neurons in an in vitro rat hippocampal slice preparation, while [D-Ala2, D-Leu5]enkephalin (DADL) was applied by perfusion at a known concentration (1 to 5 X 10-7 M), in a small droplet, or by iontophoresis into the cellular and dendritic layers of the slice. The effects of DADL on synaptic potentials and membrane properties were studied in an effort to determine the mechanisms underlying its epileptogenic action in the hippocampus. (2) DADL increased the size and often the duration of excitatory postsynaptic potentials (EPSPs) generated on either the apical or basal dendrites; this resulted in an increased discharge probability for a constant orthodromic stimulus. Extracellular field potential recordings showed a larger population spike for a given size field EPSP. These effects of DADL could be reversed substantially by perfusion with naloxone (1 to 5 X 10-7 M) and appeared qualitatively different from the epileptiform actions of penicillin. (3) DADL did not appear to increase the intrinsic excitability of the soma membrane, since membrane potential, input resistance, spike threshold, and antidromic field potentials all were unchanged. In addition, the shape of the membrane charging curve during hyperpolarizing current injection was not changed noticeably by DADL. (4) At the concentrations tested, DADL did not attenuate recurrent inhibition in the CA1 region, as evaluated by comparing in the absence and presence of DADL: (a) antidromically evoked recurrent inhibitory postsynaptic potentials (IPSPs) and their dependence of membrane potential, (b) the reduction of a synaptically driven population spike by a prior antidromic volley, (c) iontophoretic GABA (gamma-aminobutyric acid) responses. Similarly, IPSPs evoked by orthodromic stimulation appeared either unaffected or occasionally enhanced by DADL. (5) By iontophoretic mapping, it was shown that the DADL-sensitive sites are limited to stratum oriens and stratum pyramidale. Local application of DADL into stratum radiatum was relatively ineffective in enhancing the efficacy of synapses located in this region. (6) The dendritic input-output relationship between the presynaptic fiber volley and the field EPSP was not changed by DADL. This finding and the results of the iontophoretic mapping experiments suggest that increased excitatory transmitter release was not involved. (7) The data are consistent with the proposal that DADL selectively attenuates a dendritic IPSP which is virtually invisible to the soma, although the possibility cannot be ruled out that DADL may, in addition, act to enhance the responsiveness of pyramidal dendritic membrane to excitatory synaptic activation.