OBJECTIVE: Adrenal medullary chromaffin cells are known to release analgesic substances such as opioides and catecholamines. Transplantation of them is a novel method that challenges current approaches in treating chronic pain. The transplantation of xenogeneic chromaffin cells into the central nervous system(CNS) supply antinociception in animals. In this study, we investigated the analgesic effects of rat adrenal medullary chromaffin cells transplanted into the CNS of the mouse. To study the antinociceptive efficacy of transplanted chromaffin cells, the survival of rat adrenal medullary chromaffin cells transplanted into the CNS of mouse was determined. METHODS: The adrenal medullary chromaffin cells isolated from rat were transplanted into the striatum of mouse. These cells were confirmed of the release of Met-enkephalin and Leu-enkephalin by HPLC, and immunoblots for tyrosine hydroxylase(TH). Two weeks after transplantation, we performed immunohistochemistry for TH to determine the survival of implanted cells and assessed pain sensitivity at the same time. RESULTS: The isolated rat adrenal medullary chromaffin cells were positive for anti-TH antibody and released Met-enkephalin and Leu-enkephalin more than rat endothelial cells. Transplanted rat chromaffin cells were stained with anti-TH antibody in striatum of mouse after 2 weeks. Pain sensitivity was reduced on the chromaffin cell-transplanted mouse compared to endothelial cell-transplanted mouse by the hot plate test. CONCLUSION: These results suggest that the rat chromaffin cells were suitably transplanted into the CNS of mouse. This approach could be used as a therapy for reducing of chronic pain induced by cancer or neuronal injury.
Animals ; Catecholamines ; Chromaffin Cells* ; Chromatography, High Pressure Liquid ; Chronic Pain ; Endothelial Cells ; Enkephalin, Leucine ; Enkephalin, Methionine ; Enkephalins ; Immunohistochemistry ; Mice* ; Neurons ; Rats ; Tyrosine

OBJECTIVE: Using Lee et al (1996) model, we assessed the effect of opioid within the PAG on the manifestations of the neuropathic pain, and we studied the effects of naloxone on the analgesic effects of opioid. METHOD: Under pentobarbital anesthesia, male Sprague-Dawley rats were implanted with cannula in the ventral (n=10) and dorsal (n=6) PAG after the unilateral tibial and sural nerves were ligated and cut, leaving the common peroneal nerve intact. Pain sensitivity was assessed using the von Frey filament (8 mN) and acetone applied to the sensitive area for 1 week postoperatively. Rats with neuropathic pain were intracerebrally microinjected with DAMGO (0.1microgram/5microliter) and enkephaline (20microgram/5microliter) into the ventral and dorsal PAG and the pain sensitivity was assessed. Naloxone was injected to assess the observed change of pain sensitivity. RESULTS: Intracerebral microinjection of DAMGO and enkephaline into the ventral PAG, but not the dorsal PAG, increased the pain threshold which was reversed by naloxone. CONCLUSION: The results suggest that stimulation of the ventral PAG in neuropathic rats may reduce neuropathic pain via opioid-mediating pathway of the descending pain inhibition system.
Acetone ; Anesthesia ; Animals ; Catheters ; Enkephalin, Ala(2)-MePhe(4)-Gly(5)- ; Enkephalins ; Humans ; Male ; Microinjections ; Naloxone ; Neuralgia* ; Pain Threshold ; Pentobarbital ; Periaqueductal Gray* ; Peroneal Nerve ; Rats* ; Rats, Sprague-Dawley ; Sural Nerve

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

Opioid receptors have been pharmacologically classified as micro, delta, kappa and epsilon. We have recently reported that the antinociceptive effect of morphine (a micro-opioid receptor agonist), but not that of beta-endorphin (a novel micro/epsilon-opioid receptor agonist), is attenuated by whole body irradiation (WBI). It is unclear at present whether WBI has differential effects on the antinociceptive effects of micro-, delta-, kappa- and epsilon-opioid receptor agonists. In our current experiments, male ICR mice were exposed to WBI (5Gy) from a 60Co gamma-source and the antinociceptive effects of opioid receptor agonists were assessed two hours later using the hot water (52degrees C) tail-immersion test. Morphine and D-Ala2,N-Me-Phe4,Gly-olenkephalin(DAMGO), [D-Pen2-D-Pen5]enkephalin (DPDPE), trans-3,4-Dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl]-benzeneacetamide (U50,488H), and beta-endorphin were tested as agonists for micro, delta, kappa, and epsilon-opioid receptors, respectively. WBI significantly attenuated the antinociceptive effects of morphine and DAMGO, but increased those of beta-endorphin. The antinociceptive effects of DPDPE and U50,488H were not affected by WBI. In addition, to more preciously understand the differential effects of WBI on micro- and epsilon-opioid receptor agonists, we assessed pretreatment effects of beta-funaltrexamine (beta-FNA, a micro-opioid receptor antagonist) or beta-endorphin1-27 (beta-EP1-27, an epsilon-opioid receptor antagonist), and found that pretreatment with beta-FNA significantly attenuated the antinociceptive effects of morphine and beta-endorphin by WBI. beta-EP1-27 significantly reversed the attenuation of morphine by WBI and significantly attenuated the increased effects of beta-endorphin by WBI. The results demonstrate differential sensitivities of opioid receptors to WBI, especially for micro- and epsilon-opioid receptors.
3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer ; Animals ; beta-Endorphin ; Enkephalin, Ala(2)-MePhe(4)-Gly(5)- ; Enkephalin, D-Penicillamine (2,5)- ; Humans ; Male ; Mice ; Mice, Inbred ICR ; Morphine ; Naltrexone ; Receptors, Opioid ; Water ; Whole-Body Irradiation

No abstract available.
Animals ; Dynorphins* ; Enkephalins* ; Prosencephalon* ; Rats* ; RNA, Messenger*

The action of opioid on the hyperpolarization-activated cation current (Ih) in substantia gelatinosa neurons were investigated by using whole-cell voltage-clamp recording in rat spinal brain slices. Hyperpolarizing voltage steps revealed slowly activating currents in a subgroup of neurons. The half-maximal activation and the reversal potential of the current were compatible to neuronal Ih. DAMGO (1 muM), a selective-opioid agonist, reduced the amplitude of Ih reversibly. This reduction was dose-dependent and was blocked by CTOP (2 muM), a selective mu-opioid antagonist. DAMGO shifted the voltage dependence of activation to more hyperpolarized potential. Cesium (1 mM) or ZD 7288 (100 muM) blocked Ih and the currents inhibited by cesium, ZD 7288 and DAMGO shared a similar time and voltage dependence. These results suggest that activation of mu-opioid receptor by DAMGO can inhibit Ih in a subgroup of rat substantia gelatinosa neurons.
Animals ; Brain ; Cesium ; Enkephalin, Ala(2)-MePhe(4)-Gly(5)- ; Neurons* ; Rats* ; Substantia Gelatinosa*

BACKGROUND: This study was examined whether or not the orphanin FQ (OFQ)-stimulated [35S]GTPgammaS activity interact with DAMGO in the whole brain of mice. METHODS: ICR mice (male, n = 20, 20-25 g) were euthanized for the membrane preparations. In the agonist-stimulated [35S]GTPgammaS binding dose-response curves by OFQ, Ro-64-6198 and DAMGO, the EC50 (effective concentration 50, nM) and maximum stimulation (% over basal) were determined in the presence or absence of J-113397 (10 nM), a NOP (nociceptin-opioid peptide) receptor antagonist. OFQ (1micrometer), Ro-64-6198 (10micrometer), DAMGO (10micrometer) and their combination cocktail were used to determine the interaction between the NOP and MOP (micron-opioid peptide) receptor. RESULTS: The values of EC50 and maximum stimulation of [35S]GTPgammaS binding were as follows: OFQ (9.2 +/- 0.2 nM/17.9 +/- 0.1%), Ro-64-6198 (143.5 +/- 0.5 nM/18.1 +/- 0.4%), and DAMGO (680.6 +/- 0.7 nM/18.1 +/- 0.5%). J-113397 produced a 8.7 and 7.1 fold rightward shifting in the OFQ and Ro-64-6198-stimulated [35S]GTPgammaS binding dose-response curve respectively, but not in the DAMGO. OFQ combined with DAMGO-stimulated [35S]GTPgammaS binding had an additive effect, but not in the OFQ combined with Ro-64-6198. CONCLUSIONS: OFQ, Ro-64-6198 and DAMGO-stimulated [35S]GTPgammaS binding in the brain of mice has receptor selectivity. The [35S]GTPgammaS stimulation of OFQ and DAMGO had an additive effect rather than an anti-opioid effect on the level of intracellular signal transduction through agonist-stimulated [35S]GTPgammaS bindings.
Animals ; Brain* ; Enkephalin, Ala(2)-MePhe(4)-Gly(5)- ; Guanosine 5'-O-(3-Thiotriphosphate) ; Membranes ; Mice* ; Mice, Inbred ICR ; Signal Transduction

BACKGROUND: The aim of this study was to investigate the relative densities of mu-, kappa-, and delta-opioid receptors (MOR, KOR, and DOR) and [35S]GTPgammaS binding as stimulated by full agonists in mouse cerebral membranes METHODS: Naive mice (ICR, male, n = 20) were euthanized for cerebral membrane preparation. For saturation binding, [3H]DAMGO, [3H]U69593, and [3H]DPDPE were used to determine the binding parameters [Bmax (femtomoles per milligram)/Kd (nanomolar)]. And, for [35S]GTPgammaS stimulation binding, DAMGO, U69593, and SNC80 were used to determine EC50 (nanomolar) and maximum stimulation (% over basal) for MOR, KOR, and DOR, respectively. The Ke values of the corresponding selective antagonist, naloxone (20 nM), nor-BNI (3 nM), and naltrindole (3 nM) were also calculated. RESULTS: The values of Bmax and Kd for saturation binding were as follows: [3H]DAMGO (MOR; 56.4/0.92), [3H]U69593 (KOR; 23.6/1.66), and [3H]DPDPE (DOR; 71.4/3.12), and the relative proportions of MOR, KOR, and DOR were 37.3, 15.6, and 47.2%. The EC50, maximum stimulation, and the Ke value of [35S]GTPgammaS binding were as follows: DAMGO (MOR; 215.3/18.3/2.10), U69593 (KOR; 38.5/8.9/0.32), and SNC80 (DOR; 84.3/28.3/0.36). Bmax and maximum [35S]GTPgammaS binding stimulation were linearly correlated (r = 0.99). CONCLUSIONS: The densities of three opioid receptors were found to be significantly different in mouse cerebral membrane. The amount of G protein activated by agonists were found to be directly proportional to relative receptor density.
Animals ; Cerebrum ; Enkephalin, Ala(2)-MePhe(4)-Gly(5)- ; GTP-Binding Proteins* ; Humans ; Male ; Membranes* ; Mice* ; Naloxone ; Receptors, Opioid ; Specific Gravity

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