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

Objective: To explore the effects of the mu-opioid receptor (MOR) in the paraventricular nucleus (PVN) on the ejaculatory behaviors of male rats and its potential mechanisms. METHODS: Male SD rats with normal ejaculation ability were mated with female ones in hormone-induced estrus. After bilateral PVN microinjection of D-Ala-2-Me-Phe-4-Gly-ol enkephalin (DAGO) or D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2 (CTAP) with an inserted catheter, the male animals were observed for mount latency (ML), mount frequency (MF), intromission latency (IL), intromission frequency (IF), ejaculation latency (EL), ejaculation frequency (EF), post-ejaculation interval (PEI), and intromission ratio (IR). The lumbar sympathetic nerve activity (LSNA) of the rats was recorded using the PowerLab data acquisition hardware device, and the levels of norepinephrine (NE) in the peripheral plasma were measured by ELISA following microinjection of saline or different doses of DAGO or CTAP. RESULTS: Neither CTAP nor DGAO significantly affected the ML of the male rats (P > 0.05). DGAO remarkably increased IF (P < 0.01) and MF (P < 0.01), prolonged IL (P < 0.01), EL (P < 0.01) and PEI (P < 0.01), and reduced EF (P <0.01) and IR (P < 0.05). On the contrary, CTAP markedly decreased IF (P < 0.01) and MF (P < 0.01), shortened IL (P < 0.01), EL (P < 0.01) and PFI (P < 0.01), and elevated EF (P < 0.01) and IR (P < 0.01). Additionally, DAGO decreased LSNA in a dose-dependent manner and reduced the NE level in the peripheral plasma. CTAP, however, not only offset the effects of DAGO on LSNA, but also significantly increased LSNA. CONCLUSIONS MOR in PVN inhibits ejaculatory behaviors in male rats by weakening LSNA, which has provided some theoretical evidence for the use of highly selective opioids in the treatment of premature ejaculation.
Animals ; Ejaculation ; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-/pharmacology* ; Female ; Male ; Paraventricular Hypothalamic Nucleus/physiology* ; Peptide Fragments/pharmacology* ; Rats ; Rats, Sprague-Dawley ; Receptors, Opioid, mu/physiology* ; Somatostatin/pharmacology* ; Sympathetic Nervous System/physiology*

G protein-coupled receptors (GPCRs) are the largest superfamily of transmembrane receptors and have vital signaling functions in various organs. Because of their critical roles in physiology and pathology, GPCRs are the most commonly used therapeutic target. It has been suggested that GPCRs undergo massive genetic variations such as genetic polymorphisms and DNA insertions or deletions. Among these genetic variations, non-synonymous natural variations change the amino acid sequence and could thus alter GPCR functions such as expression, localization, signaling, and ligand binding, which may be involved in disease development and altered responses to GPCR-targeting drugs. Despite the clinical importance of GPCRs, studies on the genotype-phenotype relationship of GPCR natural variants have been limited to a few GPCRs such as β-adrenergic receptors and opioid receptors. Comprehensive understanding of non-synonymous natural variations within GPCRs would help to predict the unknown genotype-phenotype relationship and yet-to-be-discovered natural variants. Here, we analyzed the non-synonymous natural variants of all non-olfactory GPCRs available from a public database, UniProt. The results suggest that non-synonymous natural variations occur extensively within the GPCR superfamily especially in the N-terminus and transmembrane domains. Within the transmembrane domains, natural variations observed more frequently in the conserved residues, which leads to disruption of the receptor function. Our analysis also suggests that only few non-synonymous natural variations have been studied in efforts to link the variations with functional consequences.
Amino Acid Sequence ; DNA ; Genetic Variation ; Pathology ; Physiology ; Polymorphism, Genetic ; Receptors, Opioid ; Vital Signs

Learning/memory impairment is one of the most serious problems induced by stress, and the underlying mechanisms remain unclear. Opiates and opioid receptors are implicated in multiple physiological functions including learning and memory. However, there is no clear evidence whether the endogenous opioid system is involved in the formation of the stress-induced spatial reference memory impairment. The aim of the present study was to evaluate the role of μ opioid receptor in the stress-induced spatial reference memory impairment by means of Morris water maze (MWM) test in a mouse elevated platform stress model. The mice were trained in the MWM for four trials a session for 4 consecutive days after receiving the elevated platform stress, and intracerebroventricular injection of μ opioid receptor agonist DAMGO, antagonist CTAP or saline. Retention of the spatial training was assessed 24 h after the last training session with a 60-s free-swim probe trial using a new starting position. The results showed that intracerebroventricular injection of μ opioid receptor agonist DAMGO but not antagonist CTAP before MWM training impaired the memory retrieval of mice. Elevated platform stress before MWM training also impaired memory retrieval, which could be reversed by pre-injection of CTAP, and aggravated by DAMGO. These results suggest that endogenous opioid system may play a crucial role in the formation of the stress-induced memory impairment.
Animals ; Enkephalin, Ala(2)-MePhe(4)-Gly(5)- ; pharmacology ; Maze Learning ; Memory Disorders ; Mice ; Receptors, Opioid, mu ; physiology ; Spatial Memory ; Stress, Physiological

OBJECTIVETo investigate the effect of µ-opioid receptors (µ-ORs) in the central nucleus of the amygdala (CeA) on feeding and drinking behaviors in rats and evaluate the role of glutamate signaling in opioid-mediated ingestive behaviors.

METHODSStainless steel cannulas were implanted in the unilateral CeA for microinjection of different doses of the selective µ-OR agonist DAMGO in satiated or water-deprived male SD rats. The subsequent food intake or water intake of the rats was measured at 60, 120, and 240 min after the injection. The rats receiving microinjections of naloxone (NTX, a nonselective opioid antagonist) or D-AP-5 (a selective N-methyl-D-aspartic acid-type glutamate receptor antagonist) prior to DAMGO microinjection were tested for food intake at 60, 120, and 240 min after the injections.

RESULTSInjections of DAMGO (1-4 nmol in 0.5 µl) into the CeA significantly increased food intake in satiated rats, but did not affect water intake in rats with water deprivation. NTX (26.5 nmol in 0.5 µl) injected into the CeA antagonized DAMGO-induced feeding but D-AP-5 (6.3-25.4 nmol in 0.5 µl) injections did not produce such an effect.

CONCLUSIONµ-ORs in the CeA regulate food intake rather than water intake in rats, and the orexigenic role of µ-ORs is not dependent on the activation of the NMDA receptors in the CeA.

2-Amino-5-phosphonovalerate ; pharmacology ; Animals ; Central Amygdaloid Nucleus ; physiology ; Drinking ; physiology ; Eating ; physiology ; Enkephalin, Ala(2)-MePhe(4)-Gly(5)- ; pharmacology ; Excitatory Amino Acid Antagonists ; pharmacology ; Male ; Naloxone ; pharmacology ; Narcotic Antagonists ; pharmacology ; Rats ; Rats, Sprague-Dawley ; Receptors, Opioid, mu ; physiology

The purpose of this study is to determine 1) whether morphine postconditiong (MPostC) can attenuate the intercellular adhesion molecules-1 (ICAM-1) expression after reoxygenation injury and 2) the subtype(s) of the opioid receptors (ORs) that are involved with MPostC. Human umbilical vein endothelial cells (HUVECs) were subjected to 6 hr anoxia followed by 12 hr reoxygenation. Three morphine concentrations (0.3, 3, 30 microM) were used to evaluate the protective effect of MPostC. We also investigated blockading the OR subtypes' effects on MPostC by using three antagonists (a micro-OR antagonist naloxone, a kappa-OR antagonist nor-binaltorphimine, and a delta-OR antagonist naltrindole) and the inhibitor of protein kinase C (PKC) chelerythrine. As results, the ICAM-1 expression was significantly reduced in the MPostC (3, 30 microM) groups compared to the control group at 1, 6, 9, and 12 hours reoxygenation time. As a consequence, neutrophil adhesion was also decreased after MPostC. These effects were abolished by coadministering chelerythrine, nor-binaltorphimine or naltrindole, but not with naloxone. In conclusion, it is assumed that MPostC could attenuate the expression of ICAM-1 on endothelial cells during reoxygenation via the kappa and delta-OR (opioid receptor)-specific pathway, and this also involves a PKC-dependent pathway.
Animals ; Benzophenanthridines/pharmacology ; Endothelial Cells/cytology/*drug effects/*metabolism ; Endothelium, Vascular/cytology ; Humans ; Intercellular Adhesion Molecule-1/genetics/*metabolism ; Morphine/*pharmacology ; Naloxone/pharmacology ; Naltrexone/analogs & derivatives/pharmacology ; Narcotic Antagonists/pharmacology ; Narcotics/*pharmacology ; Protein Isoforms/metabolism ; Protein Kinase C/antagonists & inhibitors/metabolism ; Receptors, Opioid/metabolism ; Reperfusion Injury/*metabolism ; Signal Transduction/physiology ; Umbilical Veins/cytology

BACKGROUNDMu opioid receptor plays an important role in many physiological functions. Fentanyl is a widely used opioid receptor agonist for analgesia. This study was conducted to test the role of mu-opioid receptor on insulin release by determining whether fentanyl affected insulin release from freshly isolated rat pancreatic islets and if small interfering RNAs (siRNA) targeting mu-opioid receptor in the islets could knock down mu-opioid receptor expression.

METHODSIslets were isolated from ripe SD rats' pancreas by common bile duct intraductal collagenase V digestion and purified by discontinuous Ficoll density gradient centrifugation. The siRNA knock-down of mu-opioid receptor mRNA and protein in islet cells was analyzed by semi-quantitative real time-PCR and Western blotting. After siRNA-transfection for 48 hours, the islets were co-cultured with fentanyl as follows: 0 ng/ml, 3 ng/ml and 30 ng/ml for 48 hours. Then glucose-evoked insulin release was performed. As a control, the insulin release was also analyzed in islets without siRNA-trasfection after being co-cultured with fentanyl for 48 hours.

RESULTSAfter 48 hours of transfections, specific siRNA targeting of mu-opioid receptors produced significant reduction of mu-opioid receptor mRNA and protein (P < 0.01). Fentanyl significantly inhibited glucose-evoked insulin release in islets in a concentration dependent manner (P < 0.01). But after siRNA-transfection for 48 hours, the inhibition on glucose-evoked insulin release was reversed (P < 0.01).

CONCLUSIONSRNA interference specifically reduces mu-opioid receptor mRNA and protein expression, leading to reversal of the fentanyl-induced inhibition on glucose-evoked insulin release of rat islets. The activation of opioid receptor induced by fentanyl functions to inhibit insulin release. The use of RNAi presents a promising tool for future research in diabetic mechanisms and a novel therapy for diabetes.

Analgesics, Opioid ; pharmacology ; Animals ; Cell Survival ; drug effects ; Cells, Cultured ; Fentanyl ; pharmacology ; Glucose ; pharmacology ; Insulin ; secretion ; Islets of Langerhans ; drug effects ; secretion ; Male ; RNA Interference ; RNA, Messenger ; analysis ; Rats ; Rats, Sprague-Dawley ; Receptors, Opioid, mu ; antagonists & inhibitors ; genetics ; physiology

OBJECTIVEThis study aimed to investigate the possible role of Orphanin FQ (OFQ) in the regulation of hypo-thalamic gonadotropin-releasing hormone (GnRH) secretion.

METHODSThe method of push-pull perfusion and radioimmuno-assay (RIA) were adopted to examine the secretory profile of GnRH in the median eminence (ME) in freely moving ovari-ectomized (OVX) rats after intracerebroventricular (icv) injection of OFQ and/or [Nphe(1)]NC(1-13)NH(2) (NC13), a competitive antagonists of the opioid receptor-like 1 receptor (ORL1 receptor).

RESULTSGnRH release from ME significantly decreased from 40 min to 80 min after the administration of 20 and 200 nmol OFQ in OVX rats (P < 0.05). This inhibitory effect of 20 nmol OFQ could be abolished by pretreatment with equal dose of NC13. More interestingly, GnRH secretion from ME was increased markedly 60 min after icv injection of 100 and 200 nmol NC13 (P < 0.05).

CONCLUSIONOur results suggested central administration of OFQ could inhibit the release of GnRH in the ME of hypothalamus through ORL1 receptor, providing further in vivo evidence supporting the role of OFQ in the control of GnRH secretion.

Analysis of Variance ; Animals ; Dose-Response Relationship, Drug ; Female ; Gonadotropin-Releasing Hormone ; metabolism ; Median Eminence ; metabolism ; Narcotic Antagonists ; Opioid Peptides ; pharmacology ; Ovariectomy ; methods ; Peptide Fragments ; pharmacology ; Radioimmunoassay ; Rats ; Rats, Sprague-Dawley ; Receptors, Opioid ; metabolism ; Secretory Pathway ; drug effects ; Vasodilator Agents ; pharmacology ; Wakefulness ; physiology

BACKGROUNDPreconditioning with repeated electroacupuncture (EA) could mimic ischemic preconditioning to induce cerebral ischemic tolerance in rats. The present study was designed to investigate whether mu (micro)-, delta (delta)- or kappa (kappa)-opioid receptors are involved in the neuroprotection induced by repeated EA preconditioning.

METHODSThe rats were pretreated with naltrindole (NTI), nor-binaltorphimine (nor-BNI) or D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2 (CTOP), which is a highly selective delta-, kappa- or micro-opioid receptor antagonist respectively, before each EA preconditioning (30 minutes per day, 5 days). Twenty-four hours after the last EA treatment, the middle cerebral artery occlusion (MCAO) was induced for 120 minutes. The brain infarct volume was determined with 2, 3, 5-triphenyltetrazolium chloride staining at 24 hours after MCAO and compared with that in rats which only received EA preconditioning. In another experiment, the met-enkephalin-like immunoreactivity in rat brain was investigated by immunohistochemistry in both EA preconditioning and control rats.

RESULTSThe EA preconditioning reduced brain infarct volume compared with the control rats (P = 0.000). Administration of both NTI and CTOP attenuated the brain infarct volume reduction induced by EA preconditioning, presenting with larger infarct volume than that in the EA preconditioning rats (P < 0.001). But nor-BNI administration did not block the infarct volume reduction induced by EA preconditioning, presenting with smaller infarct volume than the control group rats (P = 0.000). The number of met-enkephalin-like immunoreactivity positive neurons in the EA preconditioning rats was more than that of the control rats (P = 0.000).

CONCLUSIONRepeated EA preconditioning stimulates the release of enkephalins, which may bind delta- and micro-opioid receptors to induce the tolerance against focal cerebral ischemia.

Animals ; Brain Ischemia ; prevention & control ; Electroacupuncture ; Enkephalin, Methionine ; analysis ; Immunohistochemistry ; Ischemic Preconditioning ; Male ; Naltrexone ; analogs & derivatives ; pharmacology ; Rats ; Rats, Sprague-Dawley ; Receptors, Opioid, delta ; physiology ; Receptors, Opioid, mu ; physiology ; Somatostatin ; analogs & derivatives ; pharmacology

Animals ; Cytokines ; physiology ; Humans ; Immune System ; drug effects ; physiology ; Melatonin ; pharmacology ; Opioid Peptides ; physiology ; Receptors, Melatonin ; physiology ; Stress, Physiological ; immunology ; T-Lymphocytes ; drug effects ; physiology