The study was to investigate the proliferation and apoptosis effect of nociceptin/orphanin FQ (OFQ) on 562 cells in vitro. Inhibition of K562 cells proliferation was measured by MTT assay. Morphological assessment of apoptosis was performed with Wright staining and transmission electron microscope. The apoptosis peak was measured by flow cytometry. DNA fragmentation was visualized by agarose gel electrophoresis. The results showed that OFQ time-dependently and no-dose-dependently inhibited the proliferation of K562 cells at concentrations of 10(-6) - 10(-13) mol/L. Discrete maximum of cytolytic activity was detected at concentrations of 10(-6) - 10(-7), 10(-9), 10(-12) mol/L. Compared with the control group K562 cells, the cells treated with OFQ at concentration of 10(-9) mol/L for 72 hours showed typical characteristics of apoptosis under transmission electron microscope. Apoptosis peak was found by FCM at concentration of 0, 10(-7), 10(-8), 10(-9) mol/L of OFQ for 72 hours, apoptosis rates were 0%, 22.8%, 23.8% and 26.5% respectively. DNA agarose gel electrophoresis revealed nuclear fragmentation (DNA ladder). It is concluded that OFQ can inhibit the proliferation of K562 cells and induce the apoptosis in K562 cells.
Apoptosis ; drug effects ; Cell Proliferation ; drug effects ; Humans ; K562 Cells ; Opioid Peptides ; pharmacology ; Receptors, Opioid ; agonists

BACKGROUND: Dynorphin A (1-17) is conceived as an endogenous opioid peptide with a high degree of selectivity forkappa- opioid receptor even though it has been reported to sometimes act like amicro- opioid agonist. The aim of this study was to investigate [35S] GTPgammaS binding stimulated activation by dynorphin A (1-17) in the cerebral and thalamic membranes of a rhesus monkey. METHODS: The rhesus monkey (Macaca mulatta, male, n = 1) was euthanized for the preparation of the cerebral and thalamic membranes. Protein concentrations were determined by the Bradford method. In the dynorphin A (1-17)-stimulated [35S] GTPgammaS binding dose-response curve, EC50 (effective concentration 50 nM) and maximum stimulation (% over basal) were determined in the absence or presence of themicro-andkappa-opioid receptor antagonists naloxone (20 nM) and norbinaltorphimine (nor-BNI, 3 nM), respectively. E2078-stimulated [35S] GTPgammaS binding was also determined in the absence or presence ofmicro-andkappa-opioid receptor antagonists in the cortical membrane and compared with dynorphin A (1-17). RESULTS: Values of EC50 and maximum stimulation of dynorphin A (1-17)-stimulated [35S] GTPgammaS binding were as follows: cortex (474 nM/32.0%) and thalamus (423 nM/45.3%). Nor-BNI (3 nM) did not antagonize dynorphin A (1-17)-stimulated [35S] GTPgammaS binding at all in cortical or thalamic membrane, but naloxone (20 nM) produced a 12.2 fold rightward shift of the dynorphin A (1-17)-stimulated [35S] GTPgammaS binding dose-response curve in the thalamic membrane. The EC50 and the maximum stimulation of E2078-stimulated [35S] GTPgammaS binding were 65.6 nM and 22.7%, respectively. In E2078-stimulated [35S] GTPgammaS binding, the dose-response curve was antagonized not by nor-BNI but by naloxone but in the cortical membrane (a 14.2 times rightward shift). CONCLUSIONS: Dynorphin A (1-17) is selective formicro-opioid receptor in agonist-stimulated [35S] GTPgammaS binding in the cortical and thalamic membranes of rhesus monkey.
Dynorphins* ; Guanosine 5'-O-(3-Thiotriphosphate)* ; Haplorhini* ; Humans ; Macaca mulatta ; Male ; Membranes* ; Naloxone ; Opioid Peptides ; Receptors, Opioid ; Thalamus

OBJECTIVE: To investigate whether endogenous nociceptin/orphanin FQ (N/OFQ) can inhibit arrhythmia and expression of β₁-adrenergic receptor (β₁-AR) on the surface of myocardial cell membrane in acute myocardial ischemia rats by Raf kinase inhibitory protein (RKIP). METHODS: (1) Experiment one: according to random number table method, 30 adult male Sprague-Dawley (SD) rats with only 6 weeks of age were divided into Sham group (open the chest but do not ligate the coronary artery), myocardial ischemia model group (coronary ligation of left anterior descending branch), and endogenous N/OFQ antagonists UFP-101 pretreatment group (UFP-101 group, preoperative 10 minutes after tail vein injection of 1 mL/kg UFP-101), with 10 rats in each group. Arrhythmia was recorded within 15 minutes after operation. The expression of phosphorylated RKIP (p-RKIP) was detected by Western Blot. (2) Experiment two: according to the random number table method, 30 4-week-old male SD rats were divided into UFP-101 control group, RKIP over expression group and RKIP antagonism group, with 10 rats in each group. The UFP-101 control group was intraperiton eally injected with corn oil every day, while the other two groups were injected with up adjuster of RKIP (Didymin). The rats in the three groups were all ligated after 4 weeks of feeding, and UFP-101 was injected through the tail vein 10 minutes before the operation. The RKIP antagonist group received intraperitoneal injection of the RKIP-specific antagonist locostatin 2 hours before surgery. Arrhythmia results were recorded within 15 minutes after operation. Western Blot was used to detect the expression of p-RKIP in myocardial tissue and expression of β₁-AR on the surface of myocardial cell membrane 15 minutes after surgery. RESULTS: (1) Experiment one: compared with Sham group, ventricular ectopic beat (VEB), ventricular tachycardia (VT) and ventricular fibrillation (VF) increased significantly in the model group and UFP-101 group, and arrhythmia score increased significantly. In addition, compared with the Sham group, p-RKIP expression was increased in the model group and decreased in the UFP-101 group. Compared with the model group, preconditioning with UFP-101 significantly reduced the occurrence of arrhythmia [arrhythmia score: 1.5 (0.3, 5.0) vs. 4.0 (2.0, 5.0), P < 0.05], and the expression of p-RKIP in myocardial tissue significantly decreased (p-RKIP/total RKIP: 0.20±0.11 vs. 0.43±0.11, P < 0.05). This indicated that antagonistic N/OFQ could reduce the phosphorylation of RKIP and the occurrence of arrhythmia. (2) Experiment two: compared with the UFP-101 control group, overexpression of RKIP significantly increased the occurrence of arrhythmia events, and the expression of β₁-AR on the surface of the myocardial cell membrane significantly increased. And antagonism RKIP overexpression could make the occurrence of arrhythmia eased [arrhythmia score: 3.0 (2.0, 3.0) vs. 4.0 (2.0, 5.0), P < 0.05], and significantly reduce the expression of myocardial cell membrane surface β₁-AR (β₁-AR/Na⁺-K⁺-ATPase: 0.88±0.09 vs. 1.02±0.08, P < 0.05), while there was no significant difference in total RKIP expression (total RKIP/GAPDH: 5.40±0.21 vs. 5.36±0.19, P > 0.05). This indicated that endogenous N/OFQ affected the expression of plasma β₁-AR on the surface of myocardial cell membrane and ischemic arrhythmia in rats through RKIP. CONCLUSIONS Endogenous N/OFQ can affect the expression of plasma β₁-AR on the membrane surface of ischemic myocardium and arrhythmia in rats via increased expression of RKIP phosphorylation.
Animals ; Arrhythmias, Cardiac ; Male ; Opioid Peptides/metabolism* ; Phosphatidylethanolamine Binding Protein ; Rats ; Rats, Sprague-Dawley ; Receptors, Opioid ; Nociceptin

Spinal cord stimulation (SCS)-induced analgesia was characterized, and its underlying mechanisms were examined in a spared nerve injury model of neuropathic pain in rats. The analgesic effect of SCS with moderate mechanical hypersensitivity was increased with increasing stimulation intensity between the 20% and 80% motor thresholds. Various frequencies (2, 15, 50, 100, 10000 Hz, and 2/100 Hz dense-dispersed) of SCS were similarly effective. SCS-induced analgesia was maintained without tolerance within 24 h of continuous stimulation. SCS at 2 Hz significantly increased methionine enkephalin content in the cerebrospinal fluid. The analgesic effect of 2 Hz was abolished by μ or κ opioid receptor antagonist. The effect of 100 Hz was prevented by a κ antagonist, and that of 10 kHz was blocked by any of the μ, δ, or κ receptor antagonists, suggesting that the analgesic effect of SCS at different frequencies is mediated by different endorphins and opioid receptors.
Analgesics ; Animals ; Narcotic Antagonists/pharmacology* ; Neuralgia/therapy* ; Opioid Peptides ; Rats ; Receptors, Opioid/physiology* ; Receptors, Opioid, kappa ; Spinal Cord ; Spinal Cord Stimulation

In an attempt to investigate the role of endogenous opiate in the changes of regional cerebral flow(rCBF) in intracerrebral hematoma(ICH), an experimental model of ICH was induced in the cat. Forty adult cats were divided into four groups, saline-treated normal control group(10 cats), saline-treated ICH group(10 cats) and naloxone-treated ICH group(10 cats) respectively. The ICH was induced in the right frontal region stereotactically with the autogenous arterial blood(1.5ml). The rCBF measurements done by hydrogen clearance method were carried out in each cat immediately, 20 min, 40 min, 60 min, 80 min, 100 min, 120 min, 140 min, and 160 min following ICH induction, and naloxone(10 mg/kg) was intravenously administered immediately, 60 min and 120 min after ICH induction. ICH induction resulted in increases of mean arterial blood pressure(MABP) and intracranial pressure(ICP) and decrease of rCBF of the ipsilateral hemisphere. Naloxone did not modify the pattern of MABP and ICP changes, however it transiently increased rCBF, every time the drug was administered in naloxone-treated control group and from 60 min following ICH induction in naloxone-treated ICH group. The animals were sacrificed 180 min after ICH induction and the brains were sliced and prepared with Evans Blue, which revealed no significant mass effect, midline shift or perifocal brain edema. It is concluded that the decrease of rCBF in ICH could be influenced by endogenous opiates and naloxone could improve decreased rCBF in ICH without increase of MABP or decrease of ICP.
Adult ; Animals ; Brain ; Brain Edema ; Cats* ; Evans Blue ; Hematoma* ; Humans ; Hydrogen ; Models, Theoretical ; Naloxone* ; Opioid Peptides

OBJECTIVETo explore the mechanism of electroacupuncture at "Neiguan" (PC 6) antagonizing myocardial ischemia injury.

METHODSForty rabbits were randomly divided into 4 groups, model control group, naloxone control group, electroacupuncture plus cerebral spinal fluid group, and electroacupuncture plus naloxone group, 10 rabbits in each group. The effects of electroacupuncture at "Neiguan" (PC 6) after injection of naloxone into the paraventricular nucleus (PVN) of the hypothalamus on myocardial ultrastructure, II lead ECG and transmembrane potentials of the ischemic myocardial cell in the rabbit of myocardial ischemia were investigated.

RESULTSThe injection of naloxone into PVN has no effect on myocardial functions in the normal rabbit and a little effect on ischemic myocardial functions, but at 60 min after myocardial ischemia, this has a certain protective action on myocardial functions (P < 0.05); electroacupuncture at "Neiguan" has obvious protective action on ischemic myocardium, which can be reduced by injection of naloxone into PVN (P < 0.05), but can not be blocked.

CONCLUSION(1) PVN plays an important role in the process of antagonizing myocardial ischemia by electroacupuncture at "Neiguan"; (2) endogenous opioid pepetides in PVN are involved in the acupuncture effect of antagonizing myocardial ischemia; (3) other transmitters in PVN and other nucleus groups in the center also can produce obvious effects in the antagonism to myocardial ischemia by electroacupuncture at "Neiguan".

OBJECTIVES: Research into emotional or behavioral stress typically focuses upon the hypothalamic-pituitary-adrenal axis. It is well established that the hypothalamic-pituitary-adrenocortical axis is subject to inhibitory control by opioids in a variety of animal species including pigs. Exposure to acute stress induces the upregulation of opioid receptors and the release of endogenous peptides which mediate the stress-induced analgesia. There is some literature substantiating that repeated stress can lead to changes in opioidergic neurotransmission. However, the changes are highly variable. This study was designed to observe the modulatory effect of repeated immobilization stress on opioidergic neurotransmission. METHODS: Male Sprague-Dawley rats weighing 150-200g were forced to suffer immobilization stress for 2 hours on each of 14 successive days. Then we examined the maximum binding capacity and affinity of each opioid subtypes(mu, delta, kappa). RESULTS: Repeated immobilization stress increased the binding of [3H]DPDPE on the delta-subtype opioid receptor in the striatum and hypothalamus. Saturation experiments followed by scatchard analyses of the results showed an increase in the density of delta-subtype opioid receptors, but the affinity of the delta-subtype opioid receptor remained unchanged. Repeated immobilization stress reduced enkephalin activity of striatum and hypothalamus. CONCLUSIONS: From these results, it could be concluded that repeated immobilization stress up-regulated the delta-subtype opioid receptors and reduced the activity of enkephalin, an endogenous ligand for the delta-subtype opioid receptor.
Analgesia ; Analgesics, Opioid ; Animals ; Axis, Cervical Vertebra ; Enkephalins ; Humans ; Hypothalamus ; Immobilization ; Male ; Peptides ; Rats* ; Rats, Sprague-Dawley ; Receptors, Opioid ; Swine ; Synaptic Transmission* ; Up-Regulation

BACKGROUND: Selective inhibitors of cyclooxygenase (COX)-2 are commonly used analgesics in various pain conditions. Although their actions are largely thought to be mediated by the blockade of prostaglandin (PG) biosynthesis, evidences suggesting endogenous opioid peptide link in spinal antinociception of COX inhibitor have been reported. We investigated the roles of opioid receptor subtypes in the spinal antinociception of selective COX-2 inhibitor. METHODS: To examine the antinociception of a selective COX-2 inhibitor, DUP-697 was delivered through an intrathecal catheter, 10 minutes before the formalin test in male Sprague-Dawley rats. Then, the effect of intrathecal pretreatment with CTOP, naltrindole and GNTI, which are micro, delta and kappa opioid receptor antagonist, respectively, on the analgesia induced by DUP-697 was assessed. RESULTS: Intrathecal DUP-697 reduced the flinching response evoked by formalin injection during phase 1 and 2. Naltrindole and GNTI attenuated the antinociceptive effect of intrathecal DUP-697 during both phases of the formalin test. CTOP reversed the antinociception of DUP-697 during phase 2, but not during phase 1. CONCLUSIONS: Intrathecal DUP-697, a selective COX-2 inhibitor, effectively relieved inflammatory pain in rats. The delta and kappa opioid receptors are involved in the activity of COX-2 inhibitor on the facilitated state as well as acute pain at the spinal level, whereas the micro opioid receptor is related only to facilitated pain.
Acute Pain ; Aluminum Hydroxide ; Analgesia ; Analgesics ; Animals ; Carbonates ; Catheters ; Cyclooxygenase 2 ; Formaldehyde ; Humans ; Male ; Naltrexone ; Opioid Peptides ; Pain Measurement ; Prostaglandin-Endoperoxide Synthases ; Rats ; Rats, Sprague-Dawley ; Receptors, Opioid ; Receptors, Opioid, kappa ; Somatostatin ; Thiophenes

All drugs have both favorable therapeutic and untoward adverse effects. Conventional opioid analgesics possess both analgesia and adverse reactions, such as nausea, vomiting, and respiratory depression. The opioid ligand binds to µ opioid receptor and non-selectively activates two intracellular signaling pathways: the G protein pathway induce analgesia, while the β-arrestin pathway is responsible for the opioid-related adverse reactions. An ideal opioid should activate the G protein pathway while deactivating the β-arrestin pathway. Oliceridine (TRV130) has a novel characteristic mechanism on the action of the µ receptor G protein pathway selective (µ-GPS) modulation. Even though adverse reactions (ADRs) are significantly attenuated, while the analgesic effect is augmented, the some residual ADRs persist. Consequently, a G protein biased µ opioid ligand, oliceridine, improves the therapeutic index owing to increased analgesia with decreased adverse events. This review article provides a brief history, mechanism of action, pharmacokinetics, pharmacodynamics, and ADRs of oliceridine.
Analgesia ; Analgesics, Opioid ; Animals ; Bias (Epidemiology) ; Drug-Related Side Effects and Adverse Reactions ; GTP-Binding Proteins ; Intracellular Signaling Peptides and Proteins ; Ligands ; Mice ; Mice, Knockout ; Nausea ; Patient Safety ; Pharmacokinetics ; Receptors, Opioid ; Receptors, Opioid, mu ; Respiratory Insufficiency ; Vomiting

BACKGROUND: Adrenal medullary transplants into the subarachnoid space have been demonstrated to reduce pain sensitivity. This analgesia most likely results from the release of neuroactive substances, particularly catecholamines and opioid peptides from the transplanted cells into spinal cord. METHODS: Isolated bovine chromaffin cells were encapsulated with alginate and poly-L-lysine prior to implantation into rat's subarachnoid space to protect them from host immune system. And then catecholamines from encapsulated chromaffin cells were measured quantitatively in vitro by High Performance Liquid Chromatograph. The animals were randomized into 2 groups, one of which received microencapsulated chromaffin cells and the other empty capsules. The effects of such implants were evaluated on the pain behavior resulting from a chronic constriction injury of the rat sciatic nerve for 30 days. RESULTS: Catecholamine concentration in cerebrospinal fluid (CSF) was analyzed. Data (mean SD) are considered significant at P <0.05 (ANOVA for repeated measure and Dunnett's test). Continuous release of catecholamine and met-enkephalin with responsiveness to nicotine stimulation was measured from encapsulated cells in vitro. A significant reduction of allodynic response to acetone evaporation was observed in the animals implanted with cell loaded capsules compared to control animals with empty capsules. Catecholamine concentration in CSF was higher in the cell loaded capsule group. There were no complications related to implantation. CONCLUSION: We found that encapsulated chromaffin cells released continuously catehcolamines and opioids peptides in vitro and in the CSF. Those results may prove chromaffin cell's anagesic effect indirectly.
Acetone ; Analgesia ; Analgesics, Opioid ; Animals ; Capsules ; Catecholamines* ; Cerebrospinal Fluid ; Chromaffin Cells* ; Constriction ; Drug Compounding ; Enkephalin, Methionine ; Immune System ; Nicotine ; Opioid Peptides ; Peptides ; Rats ; Sciatic Nerve ; Spinal Cord ; Subarachnoid Space