enkephalin--ala(2)-mephe(4)-gly(5)- and phenylalanyl-cyclo(cysteinyltyrosyl-tryptophyl-ornithyl-threonyl-penicillamine)threoninamide

Evidence indicates that periaqueductal gray matter (PAG) plays an important role in defensive responses and pain control. The activation of cannabinoid type-1 (CB1) or mu-opioid (MOR) receptors in the dorsal region of this structure (dPAG) inhibits fear and facilitates antinociception induced by different aversive stimuli. However, it is still unknown whether these two receptors work cooperatively in order to achieve these inhibitory actions. This study investigated the involvement and a likely interplay between CB1 and MOR receptors localized into the dPAG on the regulation of fear-like defensive responses and antinociception (evaluated in tail-flick test) evoked by dPAG chemical stimulation with N-methyl-d-aspartate (NMDA). Before the administration of NMDA, animals were first intra-dPAG injected with the CB1 agonist ACEA (0.5 pmol), or with the MOR agonist DAMGO (0.5 pmol) in combination with the respective antagonists AM251 (CB1 antagonist, 100 pmol) or CTOP (MOR antagonist, 1 nmol). To investigate the interplay between these receptors, microinjection of CTOP was combined with ACEA, or microinjection of AM251 was combined with DAMGO. Our results showed that both the intra-PAG treatments with ACEA or DAMGO inhibited NMDA-induced freezing expression, whereas only the treatment with DAMGO increased antinociception induced with NMDA, which are completely blocked by its respective antagonists. Interestingly, the inhibitory effects of ACEA or DAMGO on freezing was blocked by CTOP and AM251, respectively, indicating a functional interaction between these two receptors in the mediation of defensive behaviors. However, this cooperative interaction was not observed during the NMDA-induced antinociception. Our findings indicate that there is a cooperative action between the MOR and CB1 receptors within the dPAG and it is involved in the mediation of NMDA-induced defensive responses. Additionally, the MORs into the dPAG are involved in the modulation of the antinociceptive effects that follow a fear-like defense-reaction induced by dPAG chemical stimulation with NMDA.

Topics: Analgesics, Opioid; Animals; Arachidonic Acids; Behavior, Animal; Cannabinoid Receptor Agonists; Cannabinoid Receptor Antagonists; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Excitatory Amino Acid Agonists; Fear; Freezing Reaction, Cataleptic; Male; Microinjections; N-Methylaspartate; Nociception; Pain; Pain Measurement; Periaqueductal Gray; Piperidines; Pyrazoles; Rats; Receptor, Cannabinoid, CB1; Receptors, Opioid, mu; Somatostatin

Intrinsically photosensitive retinal ganglion cells (ipRGCs) encode light intensity and trigger reflexive responses to changes in environmental illumination. In addition to functioning as photoreceptors, ipRGCs are post-synaptic neurons in the inner retina, and there is increasing evidence that their output can be influenced by retinal neuromodulators. Here we show that opioids can modulate light-evoked ipRGC signaling, and we demonstrate that the M1, M2 and M3 types of ipRGCs are immunoreactive for μ-opioid receptors (MORs) in both mouse and rat. In the rat retina, application of the MOR-selective agonist DAMGO attenuated light-evoked firing ipRGCs in a dose-dependent manner (IC

Topics: Analgesics, Opioid; Animals; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Narcotic Antagonists; Peptides; Rats; Receptors, Opioid, mu; Retinal Ganglion Cells; Somatostatin

Mu opioid receptor activation modulates acetylcholine release in the dorsal striatum, an area deeply involved in motor function, habit formation, and reinforcement learning as well as in the pathophysiology of different movement disorders, such as dystonia. Although the role of opioids in drug reward and addiction is well established, their involvement in motor dysfunction remains largely unexplored.. We used a multidisciplinary approach to investigate the responses to mu activation in 2 mouse models of DYT1 dystonia (Tor1a. In mutant mice, selective mu receptor activation caused a stronger G-protein-dependent, dose-dependent inhibition of firing activity in cholinergic interneurons when compared with controls. In Tor1a. Mice with the DYT1 dystonia mutation exhibit an enhanced response to mu receptor activation, dependent on selective receptor gene upregulation. Our data suggest a novel role for striatal opioid signaling in motor control, and more important, identify mu opioid receptors as potential targets for pharmacological intervention in dystonia. © 2017 International Parkinson and Movement Disorder Society.

Topics: Acetylcholine; Action Potentials; Adenosine Triphosphate; Analgesics, Opioid; Animals; Calcium; Choline O-Acetyltransferase; Cholinergic Neurons; Corpus Striatum; Disease Models, Animal; Dystonia; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Gene Expression Regulation; Male; Mice; Mice, Transgenic; Molecular Chaperones; Patch-Clamp Techniques; Receptors, Opioid, mu; Somatostatin

A wealth of evidence has shown that opioid and kinin systems may control proximal defense in the dorsal periaqueductal gray matter (dPAG), a critical panic-associated area. Studies with drugs that interfere with serotonin-mediated neurotransmission suggest that the μ-opioid receptor (MOR) synergistically interacts with the 5-HT

Topics: Analgesics, Opioid; Angiotensin-Converting Enzyme Inhibitors; Animals; Anti-Anxiety Agents; Bradykinin; Bradykinin B2 Receptor Antagonists; Captopril; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Escape Reaction; Male; Panic; Periaqueductal Gray; Rats, Wistar; Receptor, Bradykinin B2; Receptors, Opioid, mu; Somatostatin

The dorsomedial hypothalamus (DMH) and the dorsal periaqueductal gray (DPAG) have been implicated in the genesis and regulation of panic-related defensive behaviors, such as escape. Previous results point to an interaction between serotonergic and opioidergic systems within the DPAG to inhibit escape, involving µ-opioid and 5-HT1A receptors (5-HT1AR). In the present study we explore this interaction in the DMH, using escape elicited by electrical stimulation of this area as a panic attack index. The obtained results show that intra-DMH administration of the non-selective opioid receptor antagonist naloxone (0.5 nmol) prevented the panicolytic-like effect of a local injection of serotonin (20 nmol). Pretreatment with the selective μ-opioid receptor (MOR) antagonist CTOP (1 nmol) blocked the panicolytic-like effect of the 5-HT1AR agonist 8-OHDPAT (8 nmol). Intra-DMH injection of the selective MOR agonist DAMGO (0.3 nmol) also inhibited escape behavior, and a previous injection of the 5-HT1AR antagonist WAY-100635 (0.37 nmol) counteracted this panicolytic-like effect. These results offer the first evidence that serotonergic and opioidergic systems work together within the DMH to inhibit panic-like behavior through an interaction between µ-opioid and 5-HT1A receptors, as previously described in the DPAG.

Topics: 8-Hydroxy-2-(di-n-propylamino)tetralin; Analgesics, Opioid; Animals; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Escape Reaction; Hypothalamus; Male; Naloxone; Panic; Panic Disorder; Periaqueductal Gray; Piperazines; Pyridines; Rats; Rats, Wistar; Receptor, Serotonin, 5-HT1A; Receptors, Opioid, mu; Serotonin; Somatostatin

We studied the rate of development of tolerance to the ethanol-induced analgesia under the effect of μ-, δ-, and κ-opioid agonists and antagonists not crossing the blood-brain barrier and rapidly inactivated by gastric and duodenal proteolytic enzymes. Activation of gastric κ-opioid receptors eliminated the analgesic effect of ethanol and accelerated the development of tolerance to ethanol-induced analgesia. In contrast, activation of gastric μ-opioid receptors decelerated the development of this tolerance. Activation of gastric δ-opioid receptors produced no effect on examined tolerance. μ-Opioid receptor antagonist decelerated and δ-opioid receptor antagonist accelerated the development of tolerance to ethanol-induced analgesia. Thus, the state of gastric opioid receptors affects the manifestation of ethanol-induced analgesia and the development of tolerance to this effect.

Topics: Analgesia; Analgesics, Opioid; Animals; Blood-Brain Barrier; Dose-Response Relationship, Drug; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, Leucine-2-Alanine; Estradiol; Ethanol; Fulvestrant; Male; Narcotic Antagonists; Pain Management; Pain Measurement; Quetiapine Fumarate; Rats; Rats, Wistar; Receptors, Opioid; Receptors, Opioid, delta; Receptors, Opioid, kappa; Receptors, Opioid, mu; Somatostatin

The nucleus accumbens shell is a key brain area mediating the reinforcing effects of ethanol (EtOH). Previously, it has been shown that the density of μ-opioid receptors in the nucleus accumbens shell is higher in alcohol-preferring Alko Alcohol (AA) rats than in alcohol-avoiding Alko Non-Alcohol rats. In addition, EtOH releases opioid peptides in the nucleus accumbens and opioid receptor antagonists are able to modify EtOH intake, all suggesting an opioidergic mechanism in the control of EtOH consumption. As the exact mechanisms of opioidergic involvement remains to be elucidated, the aim of this study was to clarify the role of accumbal μ- and κ-opioid receptors in controlling EtOH intake in alcohol-preferring AA rats.. Microinfusions of the μ-opioid receptor antagonist CTOP (0.3 and 1 μg/site), μ-opioid receptor agonist DAMGO (0.03 and 0.1 μg/site), nonselective opioid receptor agonist morphine (30 μg/site), and κ-opioid receptor agonist U50488H (0.3 and 1 μg/site) were administered via bilateral guide cannulas into the nucleus accumbens shell of AA rats that voluntarily consumed 10% EtOH solution in an intermittent, time-restricted (90-minute) 2-bottle choice access paradigm.. CTOP (1 μg/site) significantly increased EtOH intake. Conversely, DAMGO resulted in a decreasing trend in EtOH intake. Neither morphine nor U50488H had any effect on EtOH intake in the used paradigm.. The results provide further evidence for the role of accumbens shell μ-opioid receptors but not κ-opioid receptors in mediating reinforcing effects of EtOH and in regulating EtOH consumption. The results also provide support for views suggesting that the nucleus accumbens shell has a major role in mediating EtOH reward.

Topics: 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; Alcohol Drinking; Animals; Dose-Response Relationship, Drug; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Ethanol; Male; Microinjections; Morphine; Nucleus Accumbens; Rats; Receptors, Opioid, kappa; Receptors, Opioid, mu; Reward; Somatostatin; Species Specificity

Previous studies have indicated that mu-opioid receptors in the ventrolateral orbital cortex (VLO) are involved in antinociception in tail flick tests and GABAergic neurons or terminals express mu-opioid receptors in the VLO. The current study examined the effect of selective mu-opioid receptor agonist DAMGO on the GABAergic miniature inhibitory postsynaptic currents (mIPSCs) in the VLO in rats using the whole-cell patch clamp. The results demonstrated that 5 μM DAMGO application into the rat VLO slices significantly reduced the GABAergic mIPSCs frequency, without any effect on its amplitude, and this effect of DAMGO was reversed by pretreatment with selective mu-opioid receptor antagonist 1 μM CTOP. Importantly, application of CTOP alone into the VLO slices did not produce any effect on the frequency and amplitude of GABAergic mIPSCs. These results indicate a presynaptic effect of mu-opioid receptor activation on the GABAergic neurons in the VLO. The current data suggests that a presynaptic inhibition of the GABA release may contribute to the mu-opioid receptor mediated effects in the VLO and provides novel electrophysiological evidence for the underlying mechanisms of mu-opioid receptors in the VLO.

Topics: Animals; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; gamma-Aminobutyric Acid; In Vitro Techniques; Inhibitory Postsynaptic Potentials; Miniature Postsynaptic Potentials; Neurons; Patch-Clamp Techniques; Prefrontal Cortex; Rats, Sprague-Dawley; Receptors, Opioid, mu; Somatostatin

Opioid receptors can display spontaneous agonist-independent G-protein signaling (basal signaling/constitutive activity). While constitutive κ-opioid receptor (KOR) activity has been documented in vitro, it remains unknown if KORs are constitutively active in native systems. Using [(35) S] guanosine 5'-O-[gamma-thio] triphosphate coupling assay that measures receptor functional state, we identified the presence of medial prefrontal cortex KOR constitutive activity in young rats that declined with age. Furthermore, basal signaling showed an age-related decline and was insensitive to neutral opioid antagonist challenge. Collectively, the present data are first to demonstrate age-dependent alterations in the medial prefrontal cortex KOR constitutive activity in rats and changes in the constitutive activity of KORs can differentially impact KOR ligand efficacy. These data provide novel insights into the functional properties of the KOR system and warrant further consideration of KOR constitutive activity in normal and pathophysiological behavior. Opioid receptors exhibit agonist-independent constitutive activity; however, kappa-opioid receptor (KOR) constitutive activity has not been demonstrated in native systems. Our results confirm KOR constitutive activity in the medial prefrontal cortex (mPFC) that declines with age. With the ability to presynaptically inhibit multiple neurotransmitter systems in the mPFC, maturational or patho-logical alterations in constitutive activity could disrupt corticofugal glutamatergic pyramidal projection neurons mediating executive function. Regulation of KOR constitutive activity could serve as a therapeutic target to treat compromised executive function.

Topics: Age Factors; Analgesics, Opioid; Animals; Dose-Response Relationship, Drug; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Guanosine 5'-O-(3-Thiotriphosphate); Male; Naltrexone; Narcotic Antagonists; Prefrontal Cortex; Protein Binding; Radionuclide Imaging; Rats; Rats, Wistar; Receptors, Opioid, kappa; Somatostatin; Sulfur Isotopes

Previously reported results have shown that the inhibitory effect of fluoxetine on escape behavior, interpreted as a panicolytic-like effect, is blocked by pretreatment with either the opioid receptor antagonist naloxone or the 5-HT1A receptor (5-HT1A-R) antagonist WAY100635 via injection into the dorsal periaqueductal gray matter (dPAG). Additionally, reported evidence indicates that the μ-opioid receptor (MOR) interacts with the 5-HT1A-R in the dPAG. In the present work, pretreatment of the dPAG with the selective MOR blocker CTOP antagonized the anti-escape effect of chronic fluoxetine (10 mg/kg, i.p., daily, for 21 days), as measured in the elevated T-maze (ETM) test, indicating mediation of this effect by the MOR. In addition, the combined administration of sub-effective doses of the selective MOR agonist DAMGO (intra-dPAG) and sub-effective doses of chronic as well as subchronic (7 days) fluoxetine increased avoidance and escape latencies, suggesting that the activation of MORs may facilitate and accelerate the effects of fluoxetine. The current observation that MORs located in the dPAG mediate the anti-escape effect of fluoxetine may open new perspectives for the development of more efficient and fast-acting panic-alleviating drugs.

Topics: Analgesics, Opioid; Animals; Avoidance Learning; Catheters, Indwelling; Dose-Response Relationship, Drug; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Escape Reaction; Fluoxetine; Male; Microinjections; Narcotic Antagonists; Neuropsychological Tests; Periaqueductal Gray; Psychotropic Drugs; Random Allocation; Rats, Wistar; Receptors, Opioid, mu; Somatostatin

Significant opioid-dependent changes occur during the fourth postnatal week in supraspinal sites (rostroventral medulla [RVM], periaqueductal grey [PAG]) that are involved in the descending control of spinal excitability via the dorsal horn (DH). Here we report developmentally regulated changes in the opioidergic signalling within the PAG and DH, which further increase our understanding of pain processing during early life. Microinjection of the μ-opioid receptor (MOR) agonist DAMGO (30 ng) into the PAG of Sprague-Dawley rats increased spinal excitability and lowered mechanical threshold to noxious stimuli in postnatal day (P)21 rats, but had inhibitory effects in adults and lacked efficacy in P10 pups. A tonic opioidergic tone within the PAG was revealed in adult rats by intra-PAG microinjection of CTOP (120 ng, MOR antagonist), which lowered mechanical thresholds and increased spinal reflex excitability. Spinal administration of DAMGO inhibited spinal excitability in all ages, yet the magnitude of this was greater in younger animals than in adults. The expression of MOR and related peptides were also investigated using TaqMan real-time polymerase chain reaction and immunohistochemistry. We found that pro-opiomelanocortin peaked at P21 in the ventral PAG, and MOR increased significantly in the DH as the animals aged. Enkephalin mRNA transcripts preceded the increase in enkephalin immunoreactive fibres in the superficial dorsal horn from P21 onwards. These results illustrate that profound differences in the endogenous opioidergic signalling system occur throughout postnatal development.

Topics: Age Factors; Analgesics, Opioid; Analysis of Variance; Animals; Animals, Newborn; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalins; Gene Expression Regulation, Developmental; Hyperalgesia; Pain Threshold; Periaqueductal Gray; Pro-Opiomelanocortin; Rats; Rats, Sprague-Dawley; Receptors, Opioid, mu; Somatostatin; Spinal Cord

A sound strategy for improving the clinical efficacy of opioids involves exploiting positive interactions with drugs directed at other targets in pain pathways. The current study investigated the role of dopamine receptor D2 (D2R) in modulation of spinal dorsal horn excitability to noxious input, and interactions therein with μ-opioid receptor (MOR) in an animal model of neuropathic pain induced by spinal nerve ligation (SNL). C-fiber-evoked field potentials in the spinal dorsal horn were depressed concentration dependently by spinal superfusion with the D2R agonist quinpirole both in nerve-injured and sham-operated (control) rats. However, quinpirole-induced depression was significant at 10 μmol/L after SNL but only at 100 μmol/L in control rats. This quinpirole effect was completely abolished by MOR antagonist CTOP at subclinical concentration (1 μmol/L) in nerve-injured rats, but was unaltered in sham-operated rats. Nine days after SNL, D2R was upregulated to both presynaptic and postsynaptic locations in dorsal horn neurons, as revealed by double confocal immunofluorescence stainings for synaptophysin and PSD-95. In addition, D2R/MOR co-localization was increased after SNL. Co-administration of 1 μmol/L quinpirole, insufficient per se to alter evoked potentials, dramatically enhanced inhibition of evoked potentials by MOR agonist DAMGO, reducing the IC50 value of DAMGO by 2 orders of magnitude. The present data provide evidence of profound functional and subcellular changes in D2R-mediated modulation of noxious input after nerve injury, including positive interactions with spinal MOR. These results suggest D2R co-stimulation as a potential avenue to improve MOR analgesia in sustained pain states involving peripheral nerve injury.

Topics: Analgesics, Opioid; Animals; Disease Models, Animal; Disks Large Homolog 4 Protein; Dopamine Agonists; Electric Stimulation; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Evoked Potentials; Intracellular Signaling Peptides and Proteins; Male; Membrane Proteins; Nerve Fibers, Unmyelinated; Peripheral Nerve Injuries; Quinpirole; Rats; Rats, Sprague-Dawley; Receptors, Dopamine D2; Receptors, Opioid, mu; Somatostatin; Synapses; Synaptophysin; Up-Regulation

A wealth of evidence indicates that the activation of 5-HT1A and 5-HT2A receptors in the dorsal periaqueductal grey matter (dPAG) inhibits escape, a panic-related defensive behaviour. Results that were previously obtained with the elevated T-maze test of anxiety/panic suggest that 5-HT1A and μ-opioid receptors in this midbrain area work together to regulate this response. To investigate the generality of this finding, we assessed whether the same cooperative mechanism is engaged when escape is evoked by a different aversive stimulus electrical stimulation of the dPAG. Administration of the μ-receptor blocker CTOP into the dPAG did not change the escape threshold, but microinjection of the μ-receptor agonist DAMGO (0.3 and 0.5 nmol) or the 5-HT1A receptor agonist 8-OHDPAT (1.6 nmol) increased this index, indicating a panicolytic-like effect. Pretreatment with CTOP antagonised the anti-escape effect of 8-OHDPAT. Additionally, combined administration of subeffective doses of DAMGO and 8-OHDPAT increased the escape threshold, indicating drug synergism. Therefore, regardless of the aversive nature of the stimulus, μ-opioid and 5-HT1A receptors cooperatively act to regulate escape behaviour. A better comprehension of this mechanism might allow for new therapeutic strategies for panic disorder.

Topics: 8-Hydroxy-2-(di-n-propylamino)tetralin; Animals; Electric Stimulation; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Escape Reaction; Male; Microinjections; Panic; Periaqueductal Gray; Rats; Receptor, Serotonin, 5-HT1A; Receptor, Serotonin, 5-HT2A; Receptors, Opioid, mu; Somatostatin

Cholinergic interneurons (ChIs) of dorsal striatum play a key role in motor control and in behavioural learning. Neuropeptides regulate cholinergic transmission and mu opioid receptor (MOR) activation modulates striatal acetylcholine release. However, the mechanisms underlying this effect are yet uncharacterized. Here, we examined the electrophysiological responses of ChIs to the selective MOR agonist, DAMGO {[D-Ala2-MePhe4-Gly(ol)5] enkephalin}. We observed a robust, dose-dependent inhibition of spontaneous firing activity (0.06-3 μM) which was reversible upon drug washout and blocked by the selective antagonist D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2 (CTOP) (1 μM). Voltage-clamp analysis of the reversal potential of the DAMGO effect did not provide univocal results, indicating the involvement of multiple membrane conductances. The MOR-dependent effect persisted in the presence of GABAA and ionotropic glutamate receptor antagonists, ruling out an indirect effect. Additionally, it depended upon G-protein activation, as it was prevented by intrapipette GDP-β-S. Because D2 dopamine receptors (D2R) and MOR share a common post-receptor signalling pathway, occlusion experiments were performed with maximal doses of both D2R and MOR agonists. The D2R agonist quinpirole decreased spike discharge, which was further reduced by adding DAMGO. Then, D2R or MOR antagonists were used to challenge the response to the respective agonists, DAMGO or quinpirole. No cross-effect was observed, suggesting that the two receptors act independently. Our findings demonstrate a postsynaptic inhibitory modulation by MOR on ChIs excitability. Such opioidergic regulation of cholinergic transmission might contribute to shape information processing in basal ganglia circuits, and represent a potential target for pharmacological intervention.

Topics: Action Potentials; Analgesics, Opioid; Anesthetics, Local; Animals; Cadmium Chloride; Cholinergic Neurons; Corpus Striatum; Dopamine Agonists; Dose-Response Relationship, Drug; Drug Interactions; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Excitatory Amino Acid Agents; In Vitro Techniques; Male; Mice; Mice, Inbred C57BL; Neural Inhibition; Quinpirole; Receptors, Opioid, mu; Somatostatin; Tetrodotoxin

Since the discovery of opioid receptor dimers their possible roles in opioid actions were intensively investigated. Here we suggest a mechanism that may involve the μ-δ opioid heterodimers. The exact role of δ opioid receptors in antinociception and in the development of opioid tolerance is still unclear. While receptor up-regulation can be observed during the development of opioid tolerance no μ receptor down-regulation could be detected within five days. In our present work we investigated how the selective δ opioid receptor agonists and antagonists influence the antinociceptive effect of the selective μ receptor agonist DAMGO in naïve and morphine-tolerant mice. We treated male NMRI mice with 200 μmol/kg subcutaneous (s.c.) morphine twice daily for three days. On the fourth day we measured the antinociceptive effect of DAMGO alone and combined with delta ligands: DPDPE, deltorphin II (agonists), TIPP and TICPψ (antagonists), respectively, administered intrathecally (i.t.) in mouse tail-flick test. In naive control mice none of the δ ligands caused significant changes in the antinociceptive action of DAMGO. The treatment with s.c. morphine resulted in approximately four-fold tolerance to i.t. DAMGO, i.e. the ED₅₀ value of DAMGO was four times as high as in naive mice. 500 and 1000 pmol/mouse of the δ₁ selective agonist DPDPE enhanced the tolerance to DAMGO while 1000 pmol/mouse of the δ₂ selective agonist deltorphin II did not influence the degree of tolerance. However, both δ antagonists TIPP and TICPψ potentiated the antinociceptive effect of i.t. DAMGO, thus they restored the potency of DAMGO to the control level. The inhibitory action of DPDPE against the antinociceptive effect of DAMGO could be antagonized by TIPP and TICPψ. We hypothesize that during the development of morphine tolerance the formation of μδ heterodimers may contribute to the spinal opioid tolerance. δ ligands may affect the dimer formation differently. Those, like DPDPE may facilitate the dimer formation hence inhibit the antinociceptive effect of DAMGO by causing virtual μ receptor down-regulation. Ligands that do not affect the dimer formation do not influence antinociception either but ligands with the presumed capability of disconnecting the dimers may decrease the spinal tolerance to DAMGO.

Topics: Analgesics, Opioid; Animals; Disease Models, Animal; Dose-Response Relationship, Drug; Drug Administration Routes; Drug Administration Schedule; Drug Interactions; Drug Tolerance; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Ligands; Male; Mice; Morphine; Pain Measurement; Receptors, Opioid, delta; Somatostatin; Spinal Cord; Vas Deferens

Striatopallidal medium spiny neurons have been viewed as a final common path for drug reward and the ventral pallidum as an essential convergent point for hedonic and motivational signaling in the brain. The medium spiny neurons are GABAergic, but they colocalize enkephalin. Purpose of this study was to investigate the role of the opioidergic mechanisms of the ventral pallidum in ethanol self-administration behavior.. Effects of bilateral microinjections of μ-, δ-, and κ-opioid receptor agonists and antagonists into the ventral pallidum on voluntary ethanol consumption were monitored in alcohol-preferring Alko Alcohol (AA) rats using the 90-minute limited access paradigm.. Stimulation of μ-opioid receptors with DAMGO (0.01 to 0.1 μg) or morphine (1 to 10 μg) in the ventral pallidum decreased ethanol intake dose-dependently. Conversely, blocking μ-receptors with CTOP (0.3 to 3 μg) increased ethanol intake significantly. Unlike CTOP, DAMGO also increased locomotor activity. Consumption of ethanol was not modified significantly by a broad-spectrum opioid receptor antagonist naltrexone, by δ-opioid receptor agonist DPDPE or antagonist naltrindole, or by a κ-opioid receptor agonist U50,488H or antagonist nor-BNI.. The study provides evidence for μ- but not δ- or κ-opioid receptors in the ventral pallidum playing a role in the regulation of voluntary ethanol consumption. Furthermore, present findings give support to earlier work, suggesting an essential role of pallidal opioidergic transmission in drug reward.

Topics: 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; Alcohol Drinking; Analgesics, Opioid; Animals; Dose-Response Relationship, Drug; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, D-Penicillamine (2,5)-; Globus Pallidus; Male; Microinjections; Morphine; Motor Activity; Naltrexone; Narcotic Antagonists; Rats; Receptors, Opioid; Receptors, Opioid, delta; Receptors, Opioid, kappa; Receptors, Opioid, mu; Self Administration; Somatostatin

We investigated possible alterations of pharmacologically-isolated, evoked GABA(A) inhibitory postsynaptic potentials (eIPSPs) and miniature GABA(A) inhibitory postsynaptic currents (mIPSCs) in the rat central amygdala (CeA) elicited by acute application of µ-opioid receptor (MOR) agonists (DAMGO and morphine; 1 µM) and by chronic morphine treatment with morphine pellets. The acute activation of MORs decreased the amplitudes of eIPSPs, increased paired-pulse facilitation (PPF) of eIPSPs and decreased the frequency (but not the amplitude) of mIPSCs in a majority of CeA neurons, suggesting that acute MOR-dependent modulation of this GABAergic transmission is mediated predominantly via presynaptic inhibition of GABA release. We observed no significant changes in the membrane properties, eIPSPs, PPF or mIPSCs of CeA neurons during chronic morphine treatment compared to CeA of naïve or sham rats. Superfusion of the MOR antagonist CTOP (1 µM) increased the mean amplitude of eIPSPs in a majority of CeA neurons to the same degree in both naïve/sham and morphine-treated rats, suggesting a tonic activation of MORs in both conditions. Superfusion of DAMGO decreased eIPSP amplitudes and the frequency of mIPSCs equally in both naïve/sham and morphine-treated rats but decreased the amplitude of mIPSCs only in morphine treated rats, an apparent postsynaptic action. Our combined findings suggest the development of tolerance of the CeA GABAergic system to inhibitory effects of acute activation of MORs on presynaptic GABA release and possible alteration of MOR-dependent postsynaptic mechanisms that may represent important neuroadaptations of the GABAergic and MOR systems during chronic morphine treatment.

Topics: Amygdala; Analgesics, Opioid; Animals; Drug Tolerance; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Inhibitory Postsynaptic Potentials; Male; Miniature Postsynaptic Potentials; Morphine; Narcotics; Neurons; Patch-Clamp Techniques; Rats; Rats, Sprague-Dawley; Receptors, GABA-A; Receptors, Opioid, mu; Somatostatin; Synaptic Transmission

The decision to perform, or not perform, actions known to lead to a rewarding outcome is strongly influenced by the current incentive value of the reward. Incentive value is largely determined by the affective experience derived during previous consumption of the reward-the process of incentive learning. We trained rats on a two-lever, seeking-taking chain paradigm for sucrose reward, in which responding on the initial seeking lever of the chain was demonstrably controlled by the incentive value of the reward. We found that infusion of the μ-opioid receptor antagonist, CTOP (d-Phe-Cys-Tyr-d-Trp-Orn-Thr-Pen-Thr-NH(2)), into the basolateral amygdala (BLA) during posttraining, noncontingent consumption of sucrose in a novel elevated-hunger state (a positive incentive learning opportunity) blocked the encoding of incentive value information normally used to increase subsequent sucrose-seeking responses. Similar treatment with δ [N, N-diallyl-Tyr-Aib-Aib-Phe-Leu-OH (ICI 174,864)] or κ [5'-guanidinonaltrindole (GNTI)] antagonists was without effect. Interestingly, none of these drugs affected the ability of the rats to encode a decrease in incentive value resulting from experiencing the sucrose in a novel reduced-hunger state. However, the μ agonist, DAMGO ([d-Ala2, NMe-Phe4, Gly5-ol]-enkephalin), appeared to attenuate this negative incentive learning. These data suggest that upshifts and downshifts in endogenous opioid transmission in the BLA mediate the encoding of positive and negative shifts in incentive value, respectively, through actions at μ-opioid receptors, and provide insight into a mechanism through which opiates may elicit inappropriate desire resulting in their continued intake in the face of diminishing affective experience.

Topics: Amygdala; Animals; Conditioning, Operant; Drive; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, Leucine; Food; Guanidines; Male; Microinjections; Morphinans; Naltrexone; Narcotic Antagonists; Neuropsychological Tests; Rats; Rats, Long-Evans; Receptors, Opioid, kappa; Receptors, Opioid, mu; Reward; Somatostatin; Sucrose

We have previously demonstrated an opioid link in nucleus accumbens (NAc) that mediates antinociception produced by a novel ascending pain modulation pathway. For example, noxious stimulation induces heterosegmental antinociception that is mediated by both mu- and delta-opioid receptors in NAc. However, spinal intrathecal administration of the mu-receptor agonist [D-Ala(2), N-Me-Phe(4),Gly(5)-ol]-enkephalin (DAMGO) also induces heterosegmental antinociception. The aim of the present study in the rat was to identify the intra-NAc opioid receptors that mediate the antinociceptive effects of spinally administered DAMGO and also to determine the effect of NAc efferent activity on nociception. Intra-NAc administration of either the mu-opioid receptor antagonist Cys(2),Tyr(3), Orn(5),Pen(7)amide (CTOP) or the delta-opioid receptor antagonist naltrindole blocked the antinociceptive effect of spinally administered DAMGO on the jaw-opening reflex (JOR). Injection of quaternary lidocaine (QX-314) attenuated the JOR, suggesting that the output of NAc is pronociceptive. In support of this, intra-NAc injection of the excitatory amino acid agonist kainate enhanced the JOR. Thus, it is possible to modulate activity in NAc to bidirectionally attenuate or enhance nociception, suggesting a potential role for NAc in setting nociceptive sensitivity.

Topics: Analysis of Variance; Animals; Electrodes, Implanted; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Injections, Spinal; Lidocaine; Male; Naltrexone; Narcotic Antagonists; Nucleus Accumbens; Pain; Pain Measurement; Pain Perception; Rats; Rats, Sprague-Dawley; Receptors, Opioid; Somatostatin; Spinal Cord

The role of mu-opioid receptors (MORs) in the inflammatory pain processing mechanisms within the ventrobasal complex of the thalamus (VB) is not well understood. This study investigated the effect of modulating MOR activity upon nociception, by stereotaxically injecting specific ligands in the VB. Nociceptive behaviour was evaluated in two established animal models of inflammatory pain, by using the formalin (acute and tonic pain) and the ankle-bend (chronic monoarthritic pain) tests. Control (saline intra-VB injection) formalin-injected rats showed acute and tonic pain-related behaviours. In contrast, intrathalamic administration of [D-Ala(2), N-Me-Phe(4), Gly(5)-ol]-enkephalin acetate (DAMGO), a MOR-specific agonist, induced a statistically significant decrease of all tonic phase pain-related behaviours assessed until 30-35min after formalin hind paw injection. In the acute phase only the number of paw-jerks was affected. In monoarthritic rats, there was a noticeable antinociceptive effect with approximately 40min of duration, as denoted by the reduced ankle-bend scores observed after DAMGO injection. Intra-VB injection of D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH(2) (CTOP), a specific MOR antagonist, or of CTOP followed, 10min after, by DAMGO had no effects in either formalin or ankle-bend tests. Data show that DAMGO-induced MOR activation in the VB has an antinociceptive effect in the formalin test as well as in chronic pain observed in MA rats, suggesting an important and specific role for MORs in the VB processing of inflammatory pain.

Topics: Analgesics, Opioid; Analysis of Variance; Animals; Ankle; Behavior, Animal; Disease Models, Animal; Drug Interactions; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Inflammation; Male; Narcotic Antagonists; Pain; Pain Measurement; Pain Threshold; Rats; Rats, Wistar; Reaction Time; Receptors, Opioid, mu; Somatostatin; Time Factors; Ventral Thalamic Nuclei

P-type calcium channels play a key role in the synaptic transmission between mammalian central neurons since a major part of calcium entering pre-synaptic terminals is delivered via these channels. Using conventional whole-cell patch clamp techniques we have studied the effect of mu-opioids on P-type calcium channels in acutely isolated Purkinje neurons from rat cerebellum. The selective mu-opioid agonist DAMGO (10nM) produced a small, but consistent facilitation of current through P-type calcium channels (10+/-1%, n=27, p<0.001). The effect of DAMGO was rapid (less than 10s) and fully reversible. This effect was both concentration and voltage-dependent. The EC(50) for the effect of DAMGO was 1.3+/-0.4nM and the saturating concentration was 100nM. The endogenous selective agonist of mu-opioid receptors, endomorphin-1 demonstrated similar action. Intracellular perfusion of Purkinje neurons with GTPgammaS (0.5mM) or GDPbetaS (0.5mM), as well as strong depolarizing pre-pulses (+50mV), did not eliminate facilitatory action of DAMGO on P-channels indicating that this effect is not mediated by G-proteins. Furthermore, the effect of DAMGO was preserved in the presence of a non-specific inhibitor of PKA and PKC (H7, 10microM) inside the cell. DAMGO-induced facilitation of P-current was almost completely abolished by the selective mu-opioid antagonist CTOP (100nM). These observations indicate that mu-type opioid receptors modulate P-type calcium channels in Purkinje neurons via G-protein-independent mechanism.

Topics: Animals; Calcium Channels, P-Type; Cells, Cultured; Cyclic AMP-Dependent Protein Kinases; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Diphosphate; Oligopeptides; Protein Kinase C; Purkinje Cells; Rats; Receptors, Opioid, mu; Somatostatin; Thionucleotides

The depression rate of C fibre-evoked spinal field potentials by spinally applied morphine is increased in two states of spinal hyperexcitation, namely the spinal ligation model (SNL) of neuropathic pain and long-term potentiation (LTP) of C fibre-evoked spinal field potentials. This present work sought to determine opioid receptor subtypes involved in such increase in the SNL model. We recorded spinal field potentials during spinal superfusion with increasing, cumulative concentrations of selective subtype-specific agonists in rats subjected to SNL, as well as in non-ligated animals. The mu opioid receptor (MOR) agonist DAMGO significantly depressed field potentials evoked by C (100 nM) or Adelta fibres (1 microM) both in neuropathic and non-ligated rats, whereas the kappa receptor opioid (KOR) agonist +/-U-50488 was ineffective. The delta opioid receptor (DOR) (D-Ala2)-Deltorphin II was more effective in reducing C fibre-evoked spinal field potentials in rats subjected to SNL (100 nM) than in non-ligated rats (100 microM). Subclinical MOR activation (10 nM DAMGO) produced a leftward shift in (D-Ala2)-Deltorphin II dose-response curve in non-ligated rats (IC50 16.59 +/- 0.99 microM vs 120.3 +/- 1.0 microM in the absence of DAMGO), and isobolar analysis revealed synergistic interaction (interaction index 0.25). MOR blockade (100 microM CTOP) disinhibited C fibre-evoked potentials in neuropathic, but not in basal animals, and partially impeded DOR depression in both groups. DOR blockade (1 mM naltrindole) was ineffective in either group. We show that DOR-mediated depression of spinal responses to peripheral unmyelinated fibre-input is increased in the SNL model, an increase that is contributed to by positive interaction with the spinal MOR.

Topics: 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; Analgesics, Non-Narcotic; Analgesics, Opioid; Animals; Behavior, Animal; Disease Models, Animal; Dose-Response Relationship, Drug; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Evoked Potentials; Male; Narcotic Antagonists; Nerve Fibers, Unmyelinated; Neuralgia; Rats; Rats, Sprague-Dawley; Receptors, Opioid, mu; Somatostatin; Spinal Cord

The antagonism by Tyr-D-Pro-Trp-Gly-NH2 (D-Pro2-Tyr-W-MIF-1), a Tyr-Pro-Trp-Gly-NH2 (Tyr-W-MIF-1) analog, of the antinociception induced by the mu-opioid receptor agonists Tyr-W-MIF-1, [D-Ala2,NMePhe4,Gly(ol)5]-enkephalin (DAMGO), Tyr-Pro-Trp-Phe-NH2 (endomorphin-1), and Tyr-Pro-Phe-Phe-NH2 (endomorphin-2) was studied with the mouse tail-flick test. D-Pro2-Tyr-W-MIF-1 (0.5-3 nmol) given intracerebroventricularly (i.c.v.) had no effect on the thermal nociceptive threshold. High doses of D-Pro2-Tyr-W-MIF-1 (4-16 nmol) administered i.c.v. produced antinociception with a low intrinsic activity of about 30% of the maximal possible effect. D-Pro2-Tyr-W-MIF-1 (0.25-2 nmol) co-administered i.c.v. showed a dose-dependent attenuation of the antinociception induced by Tyr-W-MIF-1 or DAMGO without affecting endomorphin-2-induced antinociception. A 0.5 nmol dose of D-Pro2-Tyr-W-MIF-1 significantly attenuated Tyr-W-MIF-1-induced antinociception but not DAMGO- or endomorphin-1-induced antinociception. The highest dose (2 nmol) of D-Pro2-Tyr-W-MIF-1 almost completely attenuated Tyr-W-MIF-1-induced antinociception. However, that dose of D-Pro2-Tyr-W-MIF-1 significantly but not completely attenuated endomorphin-1 or DAMGO-induced antinociception, whereas the antinociception induced by endomorphin-2 was still not affected by D-Pro2-Tyr-W-MIF-1. Pretreatment i.c.v. with various doses of naloxonazine, a mu1-opioid receptor antagonist, attenuated the antinociception induced by Tyr-W-MIF-1, endomorphin-1, endomorphin-2, or DAMGO. Judging from the ID50 values for naloxonazine against the antinociception induced by the mu-opioid receptor agonists, the antinociceptive effect of Tyr-W-MIF-1 is extremely less sensitive to naloxonazine than that of endomorphin-1 or DAMGO. In contrast, endomorphin-2-induced antinociception is extremely sensitive to naloxonazine. The present results clearly suggest that D-Pro2-Tyr-W-MIF-1 is a selective antagonist for the mu2-opioid receptor in the mouse brain. D-Pro2-Tyr-W-MIF-1 may also discriminate between Tyr-W-MIF-1-induced antinociception and the antinociception induced by endomorphin-1 or DAMGO, which both show a preference for the mu2-opioid receptor in the brain.

Topics: Analgesics, Opioid; Animals; Brain; Disease Models, Animal; Dose-Response Relationship, Drug; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Hot Temperature; Injections, Intraventricular; Male; Mice; MSH Release-Inhibiting Hormone; Naloxone; Narcotic Antagonists; Oligopeptides; Pain; Pain Measurement; Pain Threshold; Reaction Time; Receptors, Opioid, mu; Somatostatin; Time Factors

Clinically, it has been reported that chronic pain induces depression, anxiety, and reduced quality of life. The endogenous opioid system has been implicated in nociception, anxiety, and stress. The present study was undertaken to investigate whether chronic pain could induce anxiogenic effects and changes in the opioidergic function in the amygdala in mice. We found that either injection of complete Freund's adjuvant (CFA) or neuropathic pain induced by sciatic nerve ligation produced a significant anxiogenic effect at 4 weeks after the injection or surgery. Under these conditions, the selective mu-opioid receptor agonist [D-Ala2,N-MePhe4,Gly5-ol]-enkephalin (DAMGO)- and the selective delta-opioid receptor agonist (+)-4-[(alphaR)-alpha-((2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl]-N,N-diethylbenzamide (SNC80)-stimulated [35S]GTPgammaS binding in membranes of the amygdala was significantly suppressed by CFA injection or nerve ligation. CFA injection was associated with a significant increase in the kappa-opioid receptor agonist 2-(3,4-dichlorophenyl)-N-methyl-N-[(1S)-1-phenyl-2-(1-pyrrolidinyl)ethyl]acetamide hydrochloride (ICI199,441)-stimulated [35S]GTPgammaS binding in membranes of the amygdala. The intracerebroventricular administration and microinjection of a selective mu-opioid receptor antagonist, a selective delta-opioid receptor antagonist, and the endogenous kappa-opioid receptor ligand dynorphin A caused a significant anxiogenic effect in mice. We also found that thermal hyperalgesia induced by sciatic nerve ligation was reversed at 8 weeks after surgery. In the light-dark test, the time spent in the lit compartment was not changed at 8 weeks after surgery. Collectively, the present data constitute the first evidence that chronic pain has an anxiogenic effect in mice. This phenomenon may be associated with changes in opioidergic function in the amygdala.

Topics: Amygdala; Analgesics, Opioid; Analysis of Variance; Animals; Anxiety; Behavior, Animal; Benzamides; Chronic Disease; Diazepam; Disease Models, Animal; Dose-Response Relationship, Drug; Drug Interactions; Dynorphins; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Freund's Adjuvant; Guanosine 5'-O-(3-Thiotriphosphate); Injections, Intraventricular; Male; Maze Learning; Mice; Mice, Inbred C57BL; Naltrexone; Narcotic Antagonists; Narcotics; Pain; Pain Measurement; Piperazines; Protein Binding; Pyrrolidines; Rats; Rats, Sprague-Dawley; Reaction Time; Sciatica; Somatostatin; Sulfur Isotopes; Time Factors; Tranquilizing Agents

Opioid receptors are involved in regulating neuronal survival. Here we demonstrate that activation of the mu-opioid receptor in human neuroblastoma SH-SY5Y cells led to the phosphorylations of IkappaB kinase (IKK) and p65, denoting the stimulation of the nuclear factor-kappaB (NFkappaB) transcription factor. This response was mediated through pertussis toxin-sensitive G proteins. The mu-opioid-induced IKK phosphorylation required extracellular signal-regulated protein kinase, phosphatidylinositol 3-kinase and c-Src. Moreover, c-Jun N-terminal kinase and calmodulin-dependent kinase II also participated in the IKK activation, despite the lack of involvement of phospholipase Cbeta and protein kinase C. These data suggest that the mu-opioid receptor is capable of simulating NFkappaB signaling via the phosphorylation of IKK and p65 in human neuroblastoma SH-SY5Y cells.

Topics: Analgesics, Opioid; Analysis of Variance; Blotting, Western; Cell Line, Tumor; Dose-Response Relationship, Drug; Drug Interactions; eIF-2 Kinase; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enzyme Inhibitors; Humans; Mitogen-Activated Protein Kinases; Naloxone; Narcotic Antagonists; Neuroblastoma; Pertussis Toxin; Phosphorylation; Receptors, Opioid, mu; Somatostatin; Time Factors

The effect of interactions among mu- and delta-opioid receptors, especially the putative delta(1)- and delta(2)-opioid receptors, in the nucleus accumbens on accumbal dopamine release was investigated in awake rats by in vivo brain microdialysis. In agreement with previous studies, perfusion of the nucleus accumbens with the mu-, delta(1)- and delta(2)-opioid receptor agonists [D-Ala(2),N-Me-Phe(4),Gly(5)-ol]-enkephalin (DAMGO), [D-Pen(2,5)]-enkephalin (DPDPE) and [D-Ser(2)]Leu-enkephalin-Thr(6), respectively, significantly enhanced the extracellular amount of accumbal dopamine in a dose-related manner (5.0 nmol and 50.0 nmol). However, the highest concentration tested (50.0 nmol) of DAMGO induced a biphasic effect, i.e. a rapid onset increase lasting for 75 min followed by a slower onset gradual and prolonged increase. The mu-opioid receptor antagonist D-Phe-Cys-Tyr-d-Trp-Orn-Thr-Phe-Thr-NH(2) (0.15 nmol) primarily reduced the DAMGO-induced second component. The delta(1)-opioid receptor antagonist (E)-7-benzylidenenaltrexone (0.15 nmol) significantly reduced the first component and abolished the second component induced by DAMGO, while the delta(2)-opioid receptor antagonist naltriben (1.5 nmol) significantly reduced only the first component. The DPDPE (50.0 nmol)-induced dopamine increase was almost completely abolished by (E)-7-benzylidenenaltrexone, but only partially reduced by D-Phe-Cys-Tyr-d-Trp-Orn-Thr-Phe-Thr-NH(2) and naltriben. The [D-Ser(2)]Leu-enkephalin-Thr(6) (50.0 nmol)-induced dopamine increase was almost completely abolished by naltriben, but not at all by D-Phe-Cys-Tyr-d-Trp-Orn-Thr-Phe-Thr-NH(2) and (E)-7-benzylidenenaltrexone. The non-selective opioid receptor antagonist naloxone (0.75 and 1.5 nmol) dose-dependently reduced the effects of DAMGO, DPDPE and [D-Ser(2)]Leu-enkephalin-Thr(6) but only to about 10-25% of the control values. Moreover, perfusion with the sodium channel blocker tetrodotoxin (0.1 nmol) reduced the DAMGO-induced dopamine increase by 75%, while it almost completely abolished the increase induced by DPDPE or [D-Ser(2)]Leu-enkephalin-Thr(6). The results show that stimulation of mu-opioid receptors or, to a lesser degree, delta(1)-opioid receptors results in a large naloxone-sensitive increase and a small naloxone-insensitive increase of extracellular dopamine. It is suggested that the naloxone-insensitive component is also tetrodotoxin-insensitive. Furthermore, it is hypothesized that stimulation of mu-opioid recept

Topics: Analgesics, Opioid; Anesthetics, Local; Animals; Benzylidene Compounds; Dopamine; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, D-Penicillamine (2,5)-; Enkephalin, Leucine; Extracellular Space; Male; Microdialysis; Naloxone; Naltrexone; Narcotic Antagonists; Nucleus Accumbens; Rats; Rats, Sprague-Dawley; Receptors, Opioid, delta; Receptors, Opioid, mu; Somatostatin; Tetrodotoxin

Activity-regulated cytoskeleton-associated protein (Arc) is an effector immediate early gene product implicated in long-term potentiation and other forms of neuroplasticity. Earlier studies demonstrated Arc induction in discrete brain regions by several psychoactive substances, including drugs of abuse. In the present experiments, the influence of morphine on Arc expression was assessed by quantitative reverse transcription real-time PCR and Western blotting in vivo in the mouse striatum/nucleus accumbens and, in vitro, in the mouse Neuro2A MOR1A cell line, expressing mu-opioid receptor. An acute administration of morphine produced a marked increase in Arc mRNA and protein level in the mouse striatum/nucleus accumbens complex. After prolonged opiate treatment, tolerance to the stimulatory effect of morphine on Arc expression developed. No changes in the striatal Arc mRNA levels were observed during spontaneous or opioid antagonist-precipitated morphine withdrawal. In Neuro2A MOR1A cells, acute, but not prolonged, morphine treatment elevated Arc mRNA level by activation of mu-opioid receptor. This was accompanied by a corresponding increase in Arc protein level. Inhibition experiments revealed that morphine induced Arc expression in Neuro2A MOR1A cells via intracellular signaling pathways involving mitogen-activated protein (MAP) kinases and protein kinase C. These results lend further support to the notion that stimulation of opioid receptors may exert an activating influence on some intracellular pathways and leads to induction of immediate early genes. They also demonstrate that Arc is induced in the brain in vivo after morphine administration and thus may play a role in neuroadaptations produced by the drug.

Topics: AIDS-Related Complex; Analysis of Variance; Animals; Blotting, Western; Cell Line, Tumor; Corpus Striatum; Dose-Response Relationship, Drug; Drug Administration Schedule; Drug Interactions; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enzyme Inhibitors; Flavonoids; Gene Expression; Gene Expression Regulation; Indoles; Male; Maleimides; Mice; Mice, Inbred C57BL; Morphine; Narcotics; Neuroblastoma; Receptors, Opioid, mu; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Somatostatin; Time Factors

The aim of the present study was to investigate the effect of a micro-opioid receptor agonist DAMGO (Tyr-d-Ala-Gly-NMe-Phe-Gly-ol) on the excitability of trigeminal root ganglion (TRG) neurons, projecting onto the superficial layer of the cervical dorsal horn, by using the perforated-patch technique and to determine whether TRG neurons show the expression of mRNA or functional protein for micro-opioid receptors by using reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry. TRG neurons projecting onto the superficial layer of the cervical dorsal horn were retrogradely labeled with Fluorogold (FG). The cell diameter of FG-labeled TRG neurons was small (<30 microm). Under voltage-clamp (V(h)=-60 mV), voltage-dependent K(+) currents were recorded in the TRG neurons and isolated by blocking Na(+) and Ca(2+) currents with appropriate ion replacement. Separation of the K(+) current components was achieved by the response to variation in the conditioning voltage. Two distinct K(+) current components, a transient (I(A)) and sustained (I(K)), were identified. DAMGO significantly increased I(A) by 57% (20 microM) and in a dose-dependent manner (1-50 microM). Similarly, I(K) was also enhanced by DAMGO administration (42%, 20 microM). The augmentation of both I(A) and I(K) was antagonized by a micro-opioid receptor antagonist, CTOP (d-Phe-Cys-Thr-d-Trp-Orn-Thr-Pen-Thr-NH(2)). Hyperpolarization of the membrane potential was elicited by DAMGO (20 microM) and the response was associated with a decrease in the input resistance. DAMGO induced hyperpolarization was blocked by CTOP. DAMGO-sensitive I(A) and I(K) currents were antagonized by K(+) channel blockers, 4-aminopyridine (4-AP) and tetraethylammonium (TEA). In the presence of both 4-AP and TEA, no significant changes in membrane potential induced by DAMGO application were observed. In the presence of BaCl(2), DAMGO evoked hyperpolarization with decreased resistance was observed. The firing rate of action potentials and the first spike duration induced by depolarizing step pulses were decreased in the presence of DAMGO. RT-PCR analysis demonstrated the expression of mRNA for micro-opioid receptors in the trigeminal ganglia. The micro-opioid receptor immunoreactivity was expressed in the small diameter FG-labeled TRG neurons. These results suggest that the activation of micro-opioid receptors inhibits the excitability of rat small diameter TRG neurons projecting on the superficial layer of

Topics: Analgesics, Opioid; Animals; Cervical Vertebrae; Dose-Response Relationship, Drug; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Immunohistochemistry; Male; Membrane Potentials; Neurons; Patch-Clamp Techniques; Posterior Horn Cells; Potassium Channel Blockers; Potassium Channels, Voltage-Gated; Rats; Receptors, Opioid, mu; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Somatostatin; Trigeminal Ganglion

Opioids have a selective effect on nociception with little effect on other sensory modalities. However, the cellular mechanisms for this preferential effect are not fully known. Two broad classes of nociceptors can be distinguished based on their growth factor requirements and binding to isolectin B4(IB4). In this study, we determined the difference in the modulation of voltage-gated Ca2+ currents by [D-Ala2,N-Me-Phe4,Gly-ol5]-enkephalin (DAMGO, a specific mu opioid agonist) between IB4-positive and -negative small dorsal root ganglion (DRG) neurons. Whole-cell voltage-clamp recordings were performed in acutely isolated DRG neurons in adult rats. Both 1-10 microM DAMGO and 1 to 10 microM morphine had a greater effect on high voltage-activated Ca2+ currents in IB4-negative than IB4-positive cells. However, DAMGO had no significant effect on T-type Ca2+ currents in both groups. The N-type Ca2+ current was the major subtype of Ca2+ currents inhibited by DAMGO in both IB4-positive and -negative neurons. Although DAMGO had no effect on L-type and R-type Ca2+ currents in both groups, it produced a larger inhibition on N-type and P/Q-type Ca2+ currents in IB4-negative than IB4-positive neurons. Furthermore, double labeling revealed that there was a significantly higher mu opioid receptor immunoreactivity in IB4-negative than IB4-positive cells. Thus, these data suggest that N-and P/Q-type Ca2+ currents are more sensitive to inhibition by the mu opioids in IB4-negative than IB4-positive DRG neurons. The differential sensitivity of voltage-gated Ca2+ channels to the mu opioids in subsets of DRG neurons may constitute an important analgesic mechanism of mu opioids.

Topics: Analgesics, Opioid; Animals; Calcium Channels, N-Type; Calcium Channels, P-Type; Calcium Channels, Q-Type; Dose-Response Relationship, Drug; Electrophysiology; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Ganglia, Spinal; In Vitro Techniques; Ion Channel Gating; Male; Microscopy, Confocal; Morphine; Narcotics; Patch-Clamp Techniques; Plant Lectins; Rats; Rats, Sprague-Dawley; Receptors, Opioid, mu; Somatostatin

Population spikes associated with the paired pulse ratio protocol were used to measure the presynaptic inhibition of corticostriatal transmission caused by mu-opioid receptor activation. A 1 microM of [D-Ala(2), N-MePhe(4), Gly-ol(5)]-enkephalin (DAMGO), a selective mu-opioid receptor agonist, enhanced paired pulse facilitation by 44+/-8%. This effect was completely blocked by 2 nM of the selective mu-receptor antagonist D-Phe-Cys-Tyr-D-Trp-Orn-Thr-NH (CTOP). Antagonists of N- and P/Q-type Ca(2+) channels inhibited, whereas antagonists of potassium channels enhanced, synaptic transmission. A 1 microM of omega-conotoxin GVIA, a blocker of N-type Ca(2+) channels, had no effect on the action of DAMGO, but 400 nM omega-agatoxin TK, a blocker of P/Q-type Ca(2+)-channels, partially blocked the action of this opioid. However, 5 mM Cs(2+) and 400 microM Ba(2+), unselective antagonists of potassium conductances, completely prevented the action of DAMGO on corticostriatal transmission. These data suggest that presynaptic inhibition of corticostriatal afferents by mu-opioids is mediated by the modulation of K(+) conductances in corticostriatal afferents.

Topics: Afferent Pathways; Agatoxins; Analgesics, Opioid; Animals; Barium; Calcium Channels; Cerebral Cortex; Cesium; Corpus Striatum; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; In Vitro Techniques; Male; Potassium Channels; Rats; Rats, Wistar; Receptors, Opioid, mu; Somatostatin; Spider Venoms; Synaptic Transmission

In view of the co-localization of spinal delta- and kappa-opioid receptors, we have investigated the interaction of selective opioid receptor agonists and antagonists in the spinal cord of mice in order to determine if these receptors are organized as heteromers. The finding that norbinaltorphimine (kappa) antagonized [D-Pen(2,5)]enkephalin (delta(1)), but not deltorphin II (delta(2)), strongly suggests that the putative delta(1)-subtype is a delta-kappa heteromer. Studies with selective opioid receptor (ant)agonists support this conclusion.

Topics: Animals; Benzylidene Compounds; Dynorphins; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, D-Penicillamine (2,5)-; Immune Sera; Mice; Naltrexone; Oligopeptides; Receptors, Opioid, delta; Receptors, Opioid, kappa; Receptors, Opioid, mu; Somatostatin; Spinal Cord

Immunohistochemistry and confocal microscopy were used to investigate endocytosis and recycling of the native mu opioid receptor (muOR) in enteric neurons. Isolated segments of the guinea-pig ileum were exposed to increasing concentrations of muOR agonists at 4 degrees C to allow ligand binding and warming to 37 degrees C for 0 min (baseline) to 6 h in ligand-free medium to allow receptor internalization and recycling. The endogenous ligand, [Met]enkephalin, and [D-Ala(2),MePhe(4),Gly-ol(5)] enkephalin (DAMGO), an opioid analog, and the alkaloids, etorphine and fentanyl, induced rapid internalization of muOR immunoreactivity in enteric neurons, whereas morphine did not. muOR internalization was prevented by muOR antagonists. Basal levels of muOR immunoreactivity in the cytoplasm were 10.52+/-2.05%. DAMGO (1 nM-100 microM) induced a concentration-dependent increase of muOR immunofluorescence density in the cytoplasm to a maximum of 84.37+/-2.26%. Translocation of muOR immunoreactivity in the cytoplasm was detected at 2 min, reached the maximum at 15-30 min, remained at similar levels for 2 h, began decreasing at 4 h, and was at baseline values at 6 h. A second exposure to DAMGO (100 nM) following recovery of internalized muOR immunoreactivity at the cell surface induced a translocation of muOR immunoreactivity in the cytoplasm comparable to the one observed following the first exposure (46.89+/-3.11% versus 43.31+/-3.80%). muOR internalization was prevented by hyperosmolar sucrose, phenylarsine oxide or potassium depletion, which inhibit clathrin-mediated endocytosis. muOR recycling was prevented by pre-treatment with bafilomycin A1, an acidotropic agent that inhibits endosomal acidification, but not by the protein synthesis inhibitor, cycloheximide. This study shows that native muOR in enteric neurons undergoes ligand-selective endocytosis, which is primarily clathrin-mediated, and recycles following endosomal acidification. Following recycling, muOR is activated and internalized by DAMGO indicating that recycled receptors are functional.

Topics: Analgesics, Opioid; Animals; Arsenicals; Dose-Response Relationship, Drug; Drug Interactions; Endocytosis; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalins; Enzyme Inhibitors; Guinea Pigs; Ileum; Immunohistochemistry; Ligands; Microscopy, Confocal; Naloxone; Narcotic Antagonists; Neurons; Organ Culture Techniques; Potassium; Receptors, Opioid, mu; Somatostatin; Sucrose; Time Factors

The present study investigated the effect of highly selective mu-opioid receptor (OR) agonists on lordosis behavior in ovariectomized rats treated with 3 microg of estradiol benzoate followed 48 h later by 200 microg of progesterone. Ventricular infusion of the endogenous mu-OR agonists endomorphin-1 and -2 suppressed receptive behavior in a time- and dose-dependent fashion. At 6 microg, both endomorphin-1 and -2 inhibited lordosis behavior within 30 min. However, while the effect of endomorphin-1 lasted 60 min, endomorphin-2 inhibition lasted up to 120 min after infusion. Pretreatment with naloxone (5 mg/kg sc) was able to block both endomorphin-1 and endomorphin-2 effects on lordosis. Site-specific infusions of endomorphin-1 or endomorphin-2 into the medial preoptic area (mPOA), the ventromedial nucleus of the hypothalamus (VMH), or into the mesencephalic central gray did not affect receptivity. In contrast, infusion of 1 mug of either compound into the medial septum/horizontal diagonal band of Broca inhibited lordosis in a pattern very similar to that seen after intraventricular infusions. Infusion of the potent synthetic mu-OR agonist [D-Ala(2),N-Me-Phe(4),Gly-ol(5)]-enkephalin (0.08 microg) into the VMH and mPOA inhibited lordosis behavior for at least 60 min after infusion. The nonspecific opioid receptor antagonist naloxone was able to facilitate lordosis in partially receptive female rats when infused into the mPOA but not when infused into the VMH. The behavioral effects of the agonists and antagonist used in this study suggest that the endogenous mu-opioid system modulates estrogen and progesterone-induced lordosis behavior.

Topics: Analgesics, Opioid; Animals; Brain Chemistry; Diagonal Band of Broca; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Estrogens; Female; Injections, Intraventricular; Naloxone; Narcotic Antagonists; Oligopeptides; Posture; Preoptic Area; Progesterone; Rats; Rats, Sprague-Dawley; Receptors, Opioid, mu; Sexual Behavior, Animal; Somatostatin; Ventromedial Hypothalamic Nucleus

The endomorphins are recently discovered endogenous agonists for the mu-opioid receptor (Zadina et al., 1997). Endomorphins produce analgesia; however, their role in other brain functions has not been elucidated. We have investigated the behavioral effects of endomorphin-1 in the globus pallidus, a brain region that is rich in mu-opioid receptors and involved in motor control. Bilateral administration of endomorphin-1 in the globus pallidus of rats induced orofacial dyskinesia. This effect was dose-dependent and at the highest dose tested (18 pmol per side) was sustained during the 60 min of observation, indicating that endomorphin-1 does not induce rapid desensitization of this motor response. In agreement with a lack of desensitization of mu-opioid receptors, 3 hr of continuous exposure of the cloned mu receptor to endomorphin-1 did not diminish the subsequent ability of the agonist to inhibit adenylate cyclase activity in cells expressing the cloned mu-opioid receptor. Confirming the involvement of mu-opioid receptors, the behavioral effect of endomorphin-1 in the globus pallidus was blocked by the opioid antagonist naloxone and the mu-selective peptide antagonist Cys(2)-Tyr(3)-Orn(5)-Pen(7) amide (CTOP). Furthermore, the selective mu receptor agonist [d-Ala(2)-N-Me-Phe(4)-Glycol(5)]-enkephalin (DAMGO) also stimulated orofacial dyskinesia when infused into the globus pallidus, albeit transiently. Our findings suggest that endogenous mu agonists may play a role in hyperkinetic movement disorders by inducing sustained activation of pallidal opioid receptors.

Topics: Animals; Behavior, Animal; Catalepsy; Cell Line; Colforsin; Cyclic AMP; Dose-Response Relationship, Drug; Drug Administration Routes; Dyskinesia, Drug-Induced; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Globus Pallidus; Humans; Male; Mice; Motor Activity; Naloxone; Narcotic Antagonists; Oligopeptides; Protein Binding; Rats; Rats, Sprague-Dawley; Receptors, Opioid, delta; Receptors, Opioid, kappa; Receptors, Opioid, mu; Somatostatin; Transfection

The mu-opioid receptor (MOR), a G-protein-coupled receptor, is internalized after endogenous agonist binding. Although receptor activation and internalization are separate events, internalization is a good assay for activation because endogenous opioid peptides all induce internalization. Estrogen treatment of ovariectomized rats induces MOR internalization, providing a neurochemical signature of estrogen activation of the medial preoptic nucleus. MOR activation appears to be the mechanism via which estrogen acts in the medial preoptic area to prevent the display of female reproductive behavior during the first 20-24 hr after estrogen treatment. Naltrexone, an alkaloid universal opioid receptor antagonist, prevented MOR internalization, suggesting that estrogen induces the release of endogenous opioid peptides that in turn activate the MOR. Enkephalins and beta-endorphin are nonselective endogenous MOR ligands. The most selective endogenous MOR ligands are the endomorphins. Infusions of selective MOR agonists, H-Tyr-d-Ala-Gly-N-Met-Phe-glycinol-enkephalin (DAMGO) or endomorphin-1, into the medial preoptic nucleus attenuated lordosis, and their effects were blocked with the MOR antagonist H-d-Phe-Cys-Tyr-d-Trp-Orn-Thr-Pen-Thr-NH(2) (CTOP). Infusion of endomorphin-1 internalized MOR. To determine whether progestin also acts via the MOR system to facilitate reproductive behavior, ovariectomized rats were primed with 17beta-estradiol and progesterone. Progestin facilitation of lordosis was correlated with a reduction of estrogen-induced MOR internalization. Progestin reversed estrogen-induced MOR internalization, suggesting that progesterone blocked estrogen-induced endogenous opioid release, relieving estrogen inhibition and facilitating lordosis. These results indicate a central role of MOR in the mediation of sex steroid activation of the CNS to regulate female reproductive behavior.

Topics: Analgesics, Opioid; Animals; Cell Count; Dose-Response Relationship, Drug; Drug Administration Schedule; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Estradiol; Estrogens; Female; Immunohistochemistry; Male; Naltrexone; Narcotic Antagonists; Oligopeptides; Ovariectomy; Posture; Preoptic Area; Progesterone; Rats; Rats, Long-Evans; Receptors, Opioid, mu; Sexual Behavior, Animal; Somatostatin

We investigated the ability of selective opioid agonists and antagonists to influence pro-opiomelanocortin peptide secretion from the rat neurointermediate lobe in vitro. The mu-opioid agonist DAMGO ([D-Ala(2), N-Me-Phe(4), Gly(5)-ol]enkephalin) significantly stimulated beta-endorphin and alpha-melanocyte-stimulating hormone release relative to controls early (30 min) in the incubation period. Similar effects on beta-endorphin secretion were observed with the selective mu-opioid agonist dermorphin. The delta-opioid receptor agonist DPDPE ([D-Pen(2,5)]enkephalin) weakly inhibited beta-endorphin secretion relative to controls while the kappa-opioid receptor agonist U50488 had no effect. The mu-opioid selective antagonist CTOP (D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH(2)) inhibited basal beta-endorphin secretion while kappa- and delta-opioid receptor antagonists had no effect. Our data support a role for local mu-opioid receptor control of intermediate lobe pro-opiomelanocortin peptide secretion. Peptide secretion from melanotropes appears to be tonically stimulated by activation of mu-opioid receptors in the absence of intact neuronal innervation to the intermediate lobe.

Topics: 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; alpha-MSH; Analgesics, Opioid; Animals; beta-Endorphin; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, D-Penicillamine (2,5)-; In Vitro Techniques; Male; Oligopeptides; Opioid Peptides; Pituitary Gland; Pro-Opiomelanocortin; Rats; Rats, Sprague-Dawley; Receptors, Opioid, delta; Receptors, Opioid, mu; Somatostatin

Cross-linking experiments using the (125)I-beta-endorphin revealed the presence of several receptor-related species in cell lines expressing endogenous opioid receptors, including a small molecular mass protein (approximately 22 kDa). Previous reports have suggested that this 22-kDa (125)I-beta-endorphin cross-linked protein could be the degradative product from a higher molecular mass species, i.e., a fragment of the receptor. To determine if this protein is indeed a degraded receptor fragment, (125)I-beta-endorphin was cross-linked to the (His)(6) epitope-tagged mu-opioid receptor (His-mu) stably expressed in the murine neuroblastoma Neuro(2A) cells. Similar to earlier reports with cell lines expressing endogenous receptors, two major bands of 72- and 25-kDa proteins were specifically cross-linked. Initial cross-linking experiments indicated the absolute requirement of the high-affinity (125)I-beta-endorphin binding to the mu-opioid receptor prior to the appearance of the low molecular weight species, suggesting that the 22-kDa protein could be a degraded fragment of the receptor. However, variations in the ratios of these protein bands being cross-linked by several homo- or heterobifunctional cross-linking agents were observed. Although neither the carboxyl terminus mu-opioid receptor-specific antibodies nor the antibodies against the epitope at the amino terminus of the receptor could recognize the 22-kDa protein, this (125)I-beta-endorphin cross-linked species could be coimmunoprecipitated with the receptor antibodies or could be isolated with a nickel resin affinity chromatography. The direct physical association of the 22-kDa protein with the receptor was demonstrated also by the observation that the 22-kDa protein could not bind to the nickel resin alone, but that its binding to the nickel resin was restored in the presence of the His-mu. Taken together, these results suggest that the 22-kDa protein cross-linked by (125)I-beta-endorphin is not a degradative product, but a protein located within the proximity of the mu-opioid receptor, and that it is tightly associated with the receptor.

Topics: Animals; beta-Endorphin; Cross-Linking Reagents; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Histidine; Iodine Radioisotopes; Mice; Molecular Weight; Morphine; Neuroblastoma; Proteins; Receptors, Opioid, mu; Somatostatin; Tumor Cells, Cultured

Opiate analgesics are widely used in the treatment of severe pain. Because of their importance in therapy, different strategies have been considered for making opiates more effective while curbing their liability to be abused. Although most opiates exert their analgesic effects primarily via mu opioid receptors, a number of studies have shown that delta receptor-selective drugs can enhance their potency. The molecular basis for these findings has not been elucidated previously. In the present study, we examined whether heterodimerization of mu and delta receptors could account for the cross-modulation previously observed between these two receptors. We find that co-expression of mu and delta receptors in heterologous cells followed by selective immunoprecipitation results in the isolation of mu-delta heterodimers. Treatment of these cells with extremely low doses of certain delta-selective ligands results in a significant increase in the binding of a mu receptor agonist. Similarly, treatment with mu-selective ligands results in a significant increase in the binding of a delta receptor agonist. This robust increase is also seen in SKNSH cells that endogenously express both mu and delta receptors. Furthermore, we find that a delta receptor antagonist enhances both the potency and efficacy of the mu receptor signaling; likewise a mu antagonist enhances the potency and efficacy of the delta receptor signaling. A combination of agonists (mu and delta receptor selective) also synergistically binds and potentiates signaling by activating the mu-delta heterodimer. Taken together, these studies show that heterodimers exhibit distinct ligand binding and signaling characteristics. These findings have important clinical ramifications and may provide new foundations for more effective therapies.

Topics: Analgesics, Opioid; Animals; Cell Line; CHO Cells; Cricetinae; Detergents; Dimerization; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, D-Penicillamine (2,5)-; Humans; Ligands; Mice; Narcotic Antagonists; Oligopeptides; Precipitin Tests; Receptors, Opioid, delta; Receptors, Opioid, mu; Somatostatin

The cloned delta-opioid receptor (DOR) is being investigated as a potential target for novel analgesics with an improved safety profile over mu-opioid receptor agonists such as morphine. The current study used antisense techniques to evaluate the role of DOR in mediating supraspinal antinociception in rats. All of the opioid agonists tested (delta-selective: deltorphin II, DPDPE, pCl-DPDPE, SNC80; mu-selective: DAMGO; i.c.v.) provided significant, dose-dependent antinociception in the paw pressure assay. Administration of a phosphodiester antisense oligonucleotide (i.c.v. ) targeted against DOR inhibited antinociception in response to SNC80, deltorphin II, and pCl-DPDPE compared with mismatch and saline-treated controls. However, antisense treatment did not inhibit the response to DPDPE or DAMGO. In contrast, the highly selective mu-antagonist CTOP blocked antinociception in response to ED(80) concentrations of DAMGO and DPDPE, reduced the response to pCl-DPDPE, and did not alter the response to deltorphin II or SNC80. In total, these data suggest that DOR mediates the antinociceptive response to deltorphin II, SNC80, and pCl-DPDPE at supraspinal sites and further demonstrates that the DOR-mediated response to deltorphin II and SNC80 is independent of mu-receptor activation. Conversely, supraspinal antinociception in response to DPDPE is mediated by a receptor distinct from DOR; this response is directly or indirectly sensitive to mu-receptor blockade. The distinct pharmacological profile of DPDPE suggests that either this prototypical delta-agonist mediates antinociception by a direct, nonselective interaction at mu-receptors or DPDPE interacts with a novel delta-subtype that, in turn, indirectly activates mu-receptors in the brain.

Topics: Analgesics, Opioid; Animals; Dose-Response Relationship, Drug; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, D-Penicillamine (2,5)-; Male; Oligonucleotides, Antisense; Oligopeptides; Rats; Rats, Sprague-Dawley; Receptors, Opioid, delta; Somatostatin; Spinal Cord

The cellular mechanisms underlying mu opioid facilitation of mossy fiber (MF) long-term potentiation (LTP) and synaptic transmission were investigated in the rat hippocampal slice. Naloxone (10 microM) significantly inhibited the induction of mossy fiber LTP, an effect attributed by Derrick and Martinez [B.E. Derrick, J.L.J. Martinez, Opioid receptor activation is one factor underlying the frequency dependence of mossy fiber LTP induction, J. Neurosci. 14 (1994) 4359-4367] to antagonism of endogenous opioid peptide action. We found that the inhibitory effects of naloxone were not blocked by bicuculline, suggesting that endogenous opioids did not enhance mossy fiber LTP by depressing GABAA inhibition. [d-Ala2, NMePhe4, Glyol5] enkephalin, DAMGO (300 nM), a mu opioid agonist, mimicked the action of endogenous opioids, enhancing both mossy fiber LTP induction and paired-pulse facilitation. DAMGO potentiation of the paired-pulse facilitation of mossy fiber response was also insensitive to bicuculline but was blocked by the mu selective antagonist CTOP. Further analysis of the cellular mechanism showed that the depletion of internal Ca2+ stores by thapsigargin (1 microM), or inhibition of protein kinases by application of staurosporine (1 microM) did not block the DAMGO facilitation of mossy fiber-CA3 synaptic transmission. However, application of phaclofen (100 microM GABAB receptor antagonist or SCH 50911, a more potent GABAB antagonist significantly inhibited the DAMGO effect (49+/-15%; 51+/-19% inhibition, P<0.05). The data indicate that the DAMGO effect on the mossy fiber pathway is partially mediated by a reduction in GABA activation of GABAB receptors. These findings further suggest that endogenous opioid peptides activate mu opioid receptors to facilitate mossy fiber LTP and synaptic transmission in rat hippocampus partially by GABAB receptor-mediated disinhibitory mechanism.

Topics: Analgesics, Opioid; Animals; Baclofen; Bicuculline; Electrophysiology; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalins; GABA Antagonists; Long-Term Potentiation; Male; Membrane Potentials; Mossy Fibers, Hippocampal; Naloxone; Narcotic Antagonists; Organ Culture Techniques; Rats; Rats, Sprague-Dawley; Receptors, GABA-B; Receptors, Opioid, mu; Somatostatin; Synaptic Transmission

The role of enkephalin and the opioid receptors in modulating GABA release within the rat globus pallidus (GP) was investigated using whole-cell patch recordings made from visually identified neurons. Two major GP neuronal subtypes were classified on the basis of intrinsic membrane properties, action potential characteristics, the presence of the anomalous inward rectifier (Ih), and anode break depolarizations. The mu opioid receptor agonist [D-Ala2-N-Me-Phe4-Glycol5]-enkephalin (DAMGO) (1 microM) reduced GABAA receptor-mediated IPSCs evoked by stimulation within the striatum. DAMGO also increased paired-pulse facilitation, indicative of presynaptic mu opioid receptor modulation of striatopallidal input. In contrast, the delta opioid agonist D-Pen-[D-Pen2, 5]-enkephalin (DPDPE) (1 microM) was without effect. IPSCs evoked by stimulation within the GP were depressed by application of [methionine 5']-enkephalin (met-enkephalin) (30 microM). Met-enkephalin also reduced the frequency, but not the amplitude, of miniature IPSCs (mIPSCs) and increased paired-pulse facilitation of evoked IPSCs, indicative of a presynaptic action. Both DAMGO and DPDPE reduced evoked IPSCs and the frequency, but not amplitude, of mIPSCs. However, spontaneous action potential-driven IPSCs were reduced in frequency by met-enkephalin and DAMGO, whereas DPDPE was without effect. Overall, these results indicate that presynaptic mu opioid receptors are located on striatopallidal terminals and pallidopallidal terminals of spontaneously firing GP neurons, whereas presynaptic delta opioid receptors are preferentially located on terminals of quiescent GP cells. Enkephalin, acting at both of these receptor subtypes, serves to reduce GABA release in the GP and may therefore act as an adaptive mechanism, maintaining the inhibitory function of the GP in basal ganglia circuitry.

Topics: 2-Amino-5-phosphonovalerate; 6-Cyano-7-nitroquinoxaline-2,3-dione; Analgesics; Analgesics, Opioid; Animals; Bicuculline; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, D-Penicillamine (2,5)-; Enkephalin, Methionine; Enkephalins; Excitatory Amino Acid Antagonists; GABA Antagonists; Globus Pallidus; In Vitro Techniques; Male; Membrane Potentials; Neostriatum; Neurons; Patch-Clamp Techniques; Presynaptic Terminals; Rats; Rats, Wistar; Receptors, GABA-A; Receptors, Opioid, delta; Receptors, Opioid, mu; Somatostatin; Synaptic Transmission; Tetrodotoxin

The pedunculopontine tegmental nucleus (PPTg) has been implicated in drug reward, particularly in the development of dependence. However, little is known of the receptor systems within this nucleus which might be involved. Furthermore, some research suggests that the PPTg may also be part of the neuronal circuitry involved in established drug-taking behavior.. The objective of these experiments was to examine the role of mu-opioid and nicotinic cholinergic mechanisms in the PPTg in cocaine self-administration.. Microinfusions of mu-opioid and nicotinic receptor selective compounds were made into the PPTg of rats trained to self-administer cocaine intravenously, in the vicinity of cholinergic cells which are known to project to the midbrain dopamine neurons of the ventral tegmental area (VTA).. The mu-opioid selective agonist DAMGO, tested at doses of 0, 0.05 and 0.5 microg, produced a dose-related reduction in the number of cocaine infusions obtained during the 1-h self-administration sessions. The mu-selective antagonist CTOP (0-2 microg) and nicotine (0-10 microg) did not produce significant changes in cocaine self-administration. Microinfusions of the nicotinic antagonist dihydro-beta-erythroidine (0-30 microg) produced a small but significant increase in cocaine-maintained responding.. These data show that mu-opioid mechanisms in the PPTg can influence cocaine self-administration markedly. Moreover, the data demonstrate that PPTg circuitry can influence drug reward in already-established drug-reinforced behavior, as well as during the development of dependence (as shown by previous research).

Topics: Animals; Cocaine; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalins; Male; Microinjections; Pons; Rats; Rats, Long-Evans; Receptors, Nicotinic; Receptors, Opioid, mu; Self Administration; Somatostatin; Ventral Tegmental Area

1 Although the ORL1 receptor is clearly located within the spinal cord, the functional signalling mechanism of the ORL1 receptor in the spinal cord has not been clearly documented. The present study was then to investigate the guanine nucleotide binding protein (G-protein) activation mediated through by the ORL1 receptor in the mouse spinal cord, measuring the modulation of guanosine-5'-o-(3-[35S]-thio) triphosphate ([35S]-GTPgammaS) binding by the putative endogenous ligand nociceptin, also referred as orphanin FQ. We also studied the anatomical distribution of nociceptin-like immunoreactivity and nociceptin-stimulated [35S]-GTPgammaS autoradiography in the spinal cord. 2 Immunohistochemical staining of mouse spinal cord sections revealed a dense plexus of nociceptin-like immunoreactive fibres in the superficial layers of the dorsal horn throughout the entire length of the spinal cord. In addition, networks of fibres were seen projecting from the lateral border of the dorsal horn to the lateral grey matter and around the central canal. 3 In vitro [35S]-GTPgammaS autoradiography showed high levels of nociceptin-stimulated [35S]-GTPgammaS binding in the superficial layers of the mouse dorsal horn and around the central canal, corresponding to the areas where nociceptin-like immunoreactive fibres were concentrated. 4 In [35S]-GTPgammaS membrane assay, nociceptin increased [35S]-GTPgammaS binding of mouse spinal cord membranes in a concentration-dependent and saturable manner, affording maximal stimulation of 64.1+/-2.4%. This effect was markedly inhibited by the specific ORL1 receptor antagonist [Phe1Psi (CH2-NH) Gly2] nociceptin (1 - 13) NH2. None of the mu-, delta-, and kappa-opioid and other G-protein-coupled receptor antagonists had a significant effect on basal or nociceptin-stimulated [35S]-GTPgammaS binding. 5 These findings suggest that nociceptin-containing fibres terminate in the superficial layers of the dorsal horn and the central canal and that nociceptin released in these areas may selectively stimulate the ORL1 receptor to activate G-protein. Furthermore, the unique pattern of G-protein activation in the present study provide additional evidence that nociceptin is distinct from the mu-, delta- or kappa-opioid system.

Topics: 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; Animals; Atropine; Autoradiography; Baclofen; Binding, Competitive; Dose-Response Relationship, Drug; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, D-Penicillamine (2,5)-; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Diphosphate; Haloperidol; Immunohistochemistry; In Vitro Techniques; Male; Membranes; Mice; Mice, Inbred ICR; Naltrexone; Narcotic Antagonists; Nociceptin; Nociceptin Receptor; Opioid Peptides; Peptide Fragments; Propranolol; Receptors, Opioid; Somatostatin; Spinal Cord; Sulfur Radioisotopes; Yohimbine

Activation of neurons in the midbrain periaqueductal gray (PAG) inhibits spinal dorsal horn neurons and produces behavioral antinociception in animals and analgesia in humans. Although dorsal horn regions modulated by PAG activation contain all three opioid receptor classes (mu, delta, and kappa), as well as enkephalinergic interneurons and terminal fields, descending opioid-mediated inhibition of dorsal horn neurons has not been demonstrated. We examined the contribution of dorsal horn mu-opioid receptors to the PAG-elicited descending modulation of nociceptive transmission. Single-unit extracellular recordings were made from rat sacral dorsal horn neurons activated by noxious heating of the tail. Microinjections of bicuculline (BIC) in the ventrolateral PAG led to a 60-80% decrease in the neuronal responses to heat. At the same time, the responses of the same neurons to iontophoretically applied NMDA or kainic acid were not consistently inhibited. The inhibition of heat-evoked responses by PAG BIC was reversed by iontophoretic application of the selective mu-opioid receptor antagonists, D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2 (CTOP) and D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2 (CTAP). A similar effect was produced by naloxone; however, naloxone had an excitatory influence on dorsal horn neurons in the absence of PAG-evoked descending inhibition. This is the first demonstration that endogenous opioids acting via spinal mu-opioid receptors contribute to brain stem control of nociceptive spinal dorsal horn neurons. The inhibition appears to result in part from presynaptic inhibition of afferents to dorsal horn neurons.

Topics: Adrenergic alpha-Agonists; Analgesics, Opioid; Animals; Bicuculline; Clonidine; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalins; Excitatory Amino Acid Agonists; GABA Antagonists; Hot Temperature; Iontophoresis; Kainic Acid; Male; Medulla Oblongata; N-Methylaspartate; Naloxone; Narcotic Antagonists; Nociceptors; Opioid Peptides; Pain; Peptide Fragments; Peptides; Periaqueductal Gray; Rats; Rats, Sprague-Dawley; Receptors, Opioid, mu; Somatostatin; Spinal Cord; Tail

Opioid neurons exert a tonic restraint on inhibitory VIP/PACAP/NOS motoneurons of the enteric nervous system. A decrease in opioid peptide release during the descending phase of the peristaltic reflex, which underlies propulsive activity, leads to an increase in vasoactive intestinal peptide (VIP), pituitary adenylate cyclase-activating polypeptide (PACAP), and nitric oxide (NO) release and circular muscle relaxation. These effects are accentuated by opioid receptor antagonists. Endogenous opioid peptides and selective opioid delta-, kappa- and mu-receptor agonists decreased the velocity of pellet propulsion in isolated segments of guinea pig colon, whereas selective antagonists increased velocity in a concentration-dependent fashion with an order of potency indicating preferential involvement of delta-receptors. 5-HT4 agonists (HTF-919 and R-093877), which also increase the velocity of propulsion, acted synergistically with the delta-receptor antagonist naltrindole; a threshold concentration of naltrindole (10 nM) shifted the concentration-response curve to HTF-919 to the left by 70-fold. A combination of 10 nM naltrindole with threshold concentrations of the 5-HT4 agonists caused significant increases in the velocity of propulsion (50 +/- 7 to 77 +/- 8%). We conclude that 5-HT4 agonists and opioid delta-receptor antagonists act synergistically to facilitate propulsive activity in isolated colonic segments.

Topics: Animals; Benzeneacetamides; Colon; Dynorphins; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, Leucine; Enkephalin, Methionine; Enkephalins; Guinea Pigs; In Vitro Techniques; Male; Muscle, Smooth; Naltrexone; Narcotic Antagonists; Peptide Fragments; Peristalsis; Pyrrolidines; Receptors, Opioid, delta; Receptors, Serotonin; Receptors, Serotonin, 5-HT4; Serotonin Receptor Agonists; Somatostatin

Topics: Analgesics, Opioid; Animals; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalins; Male; Mice; Mice, Inbred ICR; Nociceptors; Oligopeptides; Receptors, Opioid, mu; Somatostatin; Time Factors

Activation of 5-hydroxytryptamine-2A receptors increases the frequency of excitatory postsynaptic currents through a focal action at apical, but not basilar, dendrites of neocortical layer V pyramidal cells. Since mu-, delta- and kappa-opiate receptors are known to inhibit depolarization-induced glutamate release in cerebrocortical slices, we examined the opiate receptor subtype(s) that suppress(es) 5-hydroxytryptamine-induced excitatory postsynaptic currents in the medial prefrontal cortex and whether this suppression was occurring through a presynaptic or a postsynaptic mechanism. Only opioid agonists that act upon mu-receptors (i.e. [D-Ala2,N-Me-Phe4,Gly-ol5]enkephalin, the endogenous mu-selective agonist endomorphin-1 and the non-selective opioid agonist [Met]enkephalin) suppressed 5-hydroxytryptamine-induced excitatory postsynaptic currents. The delta-agonist [D-phen(2,5)]enkephalin and the kappa-agonist U50,488 were ineffective. Only the selective mu-antagonist CTOP blocked the suppressant effect of enkephalin, while the selective delta-antagonist naltrindole and the selective kappa-antagonist norbinaltorphimine were ineffective. Since the 5-hydroxytryptamine-induced excitatory postsynaptic currents are mediated by alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA)-type excitatory amino acid receptors, the failure of mu-agonists to either block postsynaptic AMPA responses or induce outward currents in layer V pyramidal cells suggest that mu-agonists are acting at a presynaptic site to block 5-hydroxytryptamine-induced excitatory postsynaptic currents. Strikingly, a regional selectivity in the suppressant effect of mu-receptor activation on 5-hydroxytryptamine-induced excitatory postsynaptic currents exists, as 300 nM [D-Ala2,N-Me-Phe4,Gly-ol5]enkephalin suppressed 5-hydroxytryptamine-induced excitatory postsynaptic currents in the medial prefrontal cortex by nearly 100%, while in the frontoparietal cortex 1 microM [D-Ala2,N-Me-Phe4,Gly-ol5]enkephalin suppressed 5-hydroxytryptamine-induced excitatory postsynaptic currents by only 58%. This is the first demonstration of a previously unsuspected physiological interaction between 5-hydroxytryptamine-2A and mu-opiate receptors and may be relevant to the relationship between these receptors and both mood and psychotic disorders.

Topics: alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; Animals; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, D-Penicillamine (2,5)-; Enkephalin, Methionine; Enkephalins; Evoked Potentials; Excitatory Postsynaptic Potentials; In Vitro Techniques; Male; Models, Neurological; Neocortex; Pyramidal Cells; Rats; Rats, Sprague-Dawley; Reaction Time; Receptor, Serotonin, 5-HT2A; Receptors, Opioid, mu; Receptors, Serotonin; Serotonin; Somatostatin; Synapses

The somatostatin analogues D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2 (CTOP) and D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2 (CTAP) have been used widely as selective antagonists of mu-opioid receptors. Actions of CTOP and CTAP on the membrane properties of rat locus ceruleus neurons were studied using intracellular recordings of membrane currents in superfused brain slices. CTOP increased a K+ conductance with an EC50 of 560 nM. The maximal conductance increase produced by CTOP (10 microM) was similar to that produced by high concentrations of the mu-opioid agonists D-Ala-Met-enkephalinglyol (1 microM) and Met-enkephalin (10 microM), as well as an alpha 2-adrenoceptor agonist (UK14304, 3 microM) and somatostatin (1 microM). The K+ current produced by CTOP was not antagonized by naloxone (1 microM), suggesting it was not mediated by mu-opioid receptors. The K+ currents induced by high concentrations of CTOP desensitized to 42% of the initial maximum after prolonged superfusion (t1/2 = 247 sec). In the presence of fully desensitized CTOP responses, somatostatin (1 microM) still produced near-maximal K+ currents; i.e., there was no cross-desensitization, which suggests that CTOP might act on a receptor distinct from somatostatin receptors. However, the converse did not apply; high concentrations of CTOP (30 microM) did not produce any additional current in the presence of desensitized somatostatin responses. No cross-desensitization was observed between CTOP (10-30 microM) and Met-enkephalin (30 microM) or nociceptin (3 microM) regardless of the order of drug application. Cyclo-(7-aminoheptanoyl-Phe-D-Trp-Lys-Thr[Bzl], antagonized both somatostatin-(KD = 10 microM) and CTOP-(KD = 8 microM) induced K+ currents with similar potency. Concentrations of CTOP (100 nM) that produced a small K+ current partially antagonized the actions of Met-enkephalin (10 microM) on mu-opioid receptors. In contrast to CTOP, CTAP produced no K+ current at concentrations of 300 nM and 1 microM and little current at 10 microM. CTAP potently antagonized K+ currents produced by the mu-opioid receptor agonist D-Ala-Met-enkephalin-glyol, with an equilibrium dissociation constant of 4 nM (Schild analysis). CTAP did not antagonize K+ currents produced by CTOP or somatostatin. These results demonstrate that CTOP is a potent and efficacious agonist at nonopioid receptors, whereas CTAP is a potent mu-opioid receptor antagonist with little nonopioid agonist activity in rat locus ceruleus neurons. Th

Topics: Animals; Brimonidine Tartrate; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, Methionine; Enkephalins; In Vitro Techniques; Kinetics; Locus Coeruleus; Male; Naloxone; Narcotics; Neurons; Nociceptin; Opioid Peptides; Peptide Fragments; Peptides; Potassium; Potassium Channels; Quinoxalines; Rats; Rats, Sprague-Dawley; Receptors, Opioid, mu; Somatostatin

In this paper we report on the effects of microinjections of the mu-opioid antagonist CTOP (D-Pen-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2) into the ventral tegmental area (VTA) on activity and ingestive behavior in the rat. Intra-VTA CTOP (0.015, 0.15, and 1.5 nmol per side) dose-dependently increased activity, whereas it had no effect on feeding and drinking behavior. These results are consistent with previous reports that intra-VTA injections of CTOP enhance extracellular dopamine levels in the nucleus accumbens. Furthermore, we propose a model of VTA mu-opioid mechanisms that might account for these surprising effects of intra-VTA CTOP.

Topics: Analysis of Variance; Animals; Drinking Behavior; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalins; Feeding Behavior; Microinjections; Motor Activity; Rats; Receptors, Opioid, mu; Somatostatin; Ventral Tegmental Area

Several opioid compounds were evaluated for an ability to modulate the K(+)-stimulated release of [3H]serotonin ([3H]5-hydroxytryptamine, [3H]5-HT) from rat spinal cord synaptosomal and tissue slice preparations. Selective kappa-opioid receptor agonists depressed K(+)-stimulated release of the radiolabelled transmitter from both tissue preparations, an effect which was reversed by norbinaltorphimine. Conversely, the selective mu- and delta-opioid receptor agonists [D-Ala2,NMePhe4,Gly-ol5]enkephalin (DAMGO) and [D-Pen2,D-Pen5]enkephalin (DPDPE), respectively, enhanced the K(+)-stimulated release of [3H]5-HT. This effect was only seen using the tissue slice preparation. When used at concentrations near its reported Kd for mu-opioid receptors, the selective mu-opioid receptor antagonist D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2 (CTOP) blocked the action of DAMGO, but had no effect on the action of DPDPE. However, higher concentrations of CTOP, as well as all effective concentrations of selective delta-opioid receptor antagonists, blocked the action of both DAMGO and DPDPE. All agonist effects on spinal 5-HT release, regardless of the tissue preparation, were only seen at high (microM) concentrations. Moreover, effects of the opioid agonists were not consistent with the reported involvement of spinal 5-HT neurotransmission in the mediation of their antinociceptive action. Thus, the ability of opioids to modulate spinal 5-HT release appears to be of minimal physiological significance.

Topics: Amino Acid Sequence; Analgesics; Animals; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, D-Penicillamine (2,5)-; Enkephalins; In Vitro Techniques; Isotope Labeling; Male; Molecular Sequence Data; Naltrexone; Narcotics; Potassium; Rats; Rats, Sprague-Dawley; Receptors, Opioid, delta; Receptors, Opioid, mu; Serotonin; Somatostatin; Spinal Cord; Synaptic Transmission; Synaptosomes

1. Male Sprague-Dawley rats were chronically tested with intrathecal (i.t.) receptor selective opioid antagonists to determine if antinociceptive supersensitivity developed to selective i.t. opioid receptor agonists. 2. A subcutaneously implanted osmotic minipump was used to deliver the mu-opioid receptor antagonist CTOP (0.3 nmol) or the delta-opioid receptor antagonist naltrindole (5.5 nmol) for 7 days. 3. Following a 24 hr washout period, rats received a single i.t. dose (ED50) of either DAMPGO (for CTOP-treated animals) or DPDPE (for naltrindole-treated animals) and the antinociceptive effects of the agents were tested on the tail-flick test. 4. Our findings revealed that chronic spinal treatment with selective opioid receptor antagonists did not induce an antinociceptive supersensitivity to selective opioid receptor agonists. 5. Perhaps this lack of supersensitivity is reflective of difficulties inherent to opioid receptor antagonists that do not possess negative intrinsic activity.

Topics: Animals; Dose-Response Relationship, Drug; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, D-Penicillamine (2,5)-; Enkephalins; Male; Naltrexone; Narcotic Antagonists; Rats; Rats, Sprague-Dawley; Receptors, Opioid; Somatostatin; Species Specificity; Spinal Cord

The structural basis of opioid receptors (OPRs) for the subtype-selective binding of DAMGO, a mu-opioid receptor selective ligand, was investigated using chimeric mu/kappa-OPRs. Replacement of the region from the middle of the fifth transmembrane domain to the C-terminal of mu-OPR with the corresponding region of mu-OPR remarkably decreased the binding affinity to DAMGO, while the reciprocal chimera revealed the high affinity to DAMGO. These results indicate that DAMGO distinguishes between mu- and mu-OPRs at the region around the third extracellular loop, different from the case of the distinction between mu-and delta-OPRs in which the region around the first extracellular loop is important. Furthermore, displacement studies revealed that the region around the third extracellular loop is involved in the discrimination between mu- and kappa-OPRs not only by peptidic mu- selective ligands but also by non-peptidic ligands, such as morphine and naloxone.

Topics: Amino Acid Sequence; Analgesics; Animals; Cells, Cultured; Endorphins; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalins; Ligands; Molecular Sequence Data; Morphine; Naloxone; Oligopeptides; Opioid Peptides; Receptors, Opioid; Receptors, Opioid, delta; Receptors, Opioid, kappa; Receptors, Opioid, mu; Recombinant Fusion Proteins; Somatostatin; Structure-Activity Relationship

Mastoparan activated in a concentration-dependent manner the low Km GTPase activity in P2 fractions from mouse periaquedultal grey matter (PAG). This peptide at 1-10 mM produced increases of 30-70% over the basal value of 90-120 pmol Pi/mg/min. A series of substances displaying antagonist activity at cellular receptors and not modifying the GTPase function, when used at nanomolar and micromolar concentrations enhanced the effect of mastoparan upon this enzyme. These included antagonists of receptors coupling G proteins: naloxone (non selective opioid antagonist), CTOP (m opioid receptors), ICI 174,864 (d opioid receptors), nor-BNI (k opioid receptors), sulpiride (D2 dopaminergic antagonist), idazoxan (a2 adrenergic antagonist). Bicuculline, antagonist of a receptor not linked to G proteins, GABAA, did not alter the effect of mastoparan on the GTPase. The m opioid agonist, DAMGO, prevented naloxone from increasing the function of the mastoparan-activated enzyme. Thus, mastoparan appears to act on Gi/Go proteins at a site not directly related to the receptor binding domain.

Topics: Animals; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalins; Enzyme Activation; GTP Phosphohydrolases; GTP-Binding Proteins; In Vitro Techniques; Intercellular Signaling Peptides and Proteins; Kinetics; Male; Mice; Naloxone; Peptides; Periaqueductal Gray; Receptors, Cell Surface; Somatostatin; Wasp Venoms

Brain regions that may be functionally involved in the neuropharmacological actions of mu-opioid agonists have been examined in conscious rats using the quantitative [14C]2-deoxyglucose autoradiographic technique. At 0.5 microgram and 1 microgram intracerebroventricularly the highly selective mu-opioid receptor agonist D-Ala2, MePhe4, Gly-ol5-enkephalin effected statistically significant increases as well as statistically significant decreases in regional glucose utilization: in limbic structures, such as hippocampal formation, medial amygdala and lateral septum, glucose utilization was most prominently increased after D-Ala2, MePhe4, Gly-ol5-enkephalin; glucose utilization was further increased in the lateral habenular nucleus, the hypothalamus, ventromedial nucleus and dorsal raphe; whereas decreases were found in the mamillary body and anterior thalamus. Glucose utilization in structures associated with somatosensory and nociceptive processing was increased in the central gray of the midbrain and decreased in the nucleus gelatinosus. Only increases in glucose utilization were produced by D-Ala2; MePhe4, Gly-ol5-enkephalin in brain regions involved in motor control, including the globus pallidus, the substantia nigra, pars reticulata, the nucleus ruber and the cerebellum, and brain regions involved in visual processing--the visual cortex and superior colliculus deep layer. It is concluded that this pattern of regional changes underlies the mu-opioid receptor-mediated antinociceptive-, epileptogenic-, memory- and mood-modulating actions of mu-opioid agonists.

Topics: Amino Acid Sequence; Animals; Autoradiography; Brain; Carbon Dioxide; Deoxyglucose; Dose-Response Relationship, Drug; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalins; Glucose; Injections, Intraventricular; Male; Molecular Sequence Data; Oxygen; Partial Pressure; Rats; Rats, Sprague-Dawley; Receptors, Opioid, mu; Somatostatin

The irreversible opioid receptor antagonists naloxonazine and beta-funaltrexamine have been used to determine whether multiple mu-opioid receptors exist on undifferentiated SH-SY5Y human neuroblastoma cells. Naloxonazine binds irreversibly to the mu 1-opioid receptor subtype and reversibly to the mu 2-opioid receptor subtype. On SH-SY5Y cells naloxonazine afforded a Ki of 3.4 +/- 0.7 nM, and was fully reversible, indicating the mu-opioid receptor population on SH-SY5Y cells was solely of the mu 2-opioid receptor subtype. The alkylating agent beta-funaltrexamine was maximally able to alkylate only 60% of the mu-opioid receptor sites on SH-SY5Y cells, labelled with [3H]diprenorphine or [3H][D-Ala2,MePhe4,Gly(ol)5]enkephalin (DAMGO). The reversible binding of naloxonazine and the insensitivity of a percentage of the mu-opioid receptor sites to alkylation by beta-funaltrexamine suggests that differences do exist in the mu 2-opioid receptor population on undifferentiated SH-SY5Y cells. This may indicate further heterogeneity or the inability of beta-funaltrexamine to alkylate all relevant nucleophilic groups in a single population of receptors.

Topics: Amino Acid Sequence; Animals; Brain Neoplasms; Cerebral Cortex; Diprenorphine; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalins; Humans; Ligands; Molecular Sequence Data; Naloxone; Naltrexone; Neuroblastoma; Rats; Receptors, Opioid, mu; Somatostatin; Tumor Cells, Cultured

This study examined the influence of acute and repeated administration of the kappa-opioid receptor antagonist, nor-binaltorphimine, upon opioid-induced antinociception as measured by the tail-pressure test. A single intracerebroventricular (i.c.v.) injection of nor-binaltorphimine (30 micrograms) administered 1, 10 or 30 days prior to algesiometric testing prevented the analgesic effect of the kappa-opioid receptor agonist, (5 alpha, 7 alpha, 8 beta)-(-)-N- methyl-N-(7-(1-pyrrolidinyl)-1-oxaspiro(4,5)dec-8-yl)benzenacet amide (U69593). The analgesic effect of the mu-opioid receptor agonist, [D-Ala2,N-methyl-Phe4,Gly5-ol]enkephalin (DAMGO), and the delta-opioid receptor agonist, [D-Pen2,D-Pen5]enkephalin (DPDPE), was not modified. In contrast, when nor-binaltorphimine was administered repeatedly (twice daily i.c.v. administration of 30 micrograms nor-binaltorphimine for 10 days), the analgesic effect of DAMGO, DPDPE as well as U69593 was abolished. In the case of mu- and delta-opioid receptor agonists, this abolition was apparent when testing occurred 1 or 2, but not 5 days after termination of nor-binaltorphimine treatment. This treatment regimen also resulted in a long-lasting antagonism (e.g. 20 days) of U69593-induced analgesia. These data show that, depending on the treatment regimen employed, nor-binaltorphimine can function as a selective kappa-opioid receptor antagonist, or as an antagonist at multiple opioid receptor subtypes. Further, they demonstrate that nor-binaltorphimine functions as a long-lasting kappa-opioid receptor antagonist in vivo.

Topics: Amino Acid Sequence; Analgesics; Animals; Benzeneacetamides; Drug Interactions; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, D-Penicillamine (2,5)-; Enkephalin, Leucine; Enkephalins; Injections, Intraventricular; Male; Molecular Sequence Data; Naltrexone; Narcotic Antagonists; Pain; Pain Threshold; Pyrrolidines; Rats; Rats, Sprague-Dawley; Receptors, Opioid, kappa; Somatostatin

Intracellular recordings were made from layer V pyramidal neurons in slices of rat anterior cingulate cortex, using electrodes that contained potassium methylsulfate and biocytin. [Met5]enkephalin (300 nM to 30 microM) reversibly reduced the amplitude of EPSPs evoked by stimulation of the subcortical white matter; the half-maximal concentration was about 800 nM. These EPSPs were blocked by (+/-)-2-amino-5-phosphonovaleric acid and 6-cyano-7-nitroquinoxaline-2,3-dione. [Met5]enkephalin also reduced the amplitude of bicuculline-sensitive IPSPs evoked by stimulation within layer V; the half-maximal concentration was about 60 nM. Both these actions of [Met5]enkephalin were mimicked by the delta-selective agonist DPDPE (Tyr-D-Pen-Gly-Phe-D-Pen) but not by the mu-selective agonist DAMGO (Tyr-D-Ala-Gly-MePhe-Gly-ol); they were blocked by the delta-selective antagonist naltrindole (apparent dissociation constant of about 0.3 nM) but not by the mu-selective antagonist CTOP (D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2). [Met5]enkephalin did not change the amplitudes of depolarizations evoked by direct application of glutamate or hyperpolarizations evoked by direct application of muscimol (at -55 mV). Fifty percent (22 of 45) of pyramidal cells were hyperpolarized by [Met5]enkephalin; this resulted from an increase in potassium conductance, and it was mimicked by DPDPE and blocked by naltrindole. Five of seven nonpyramidal cells were hyperpolarized by [Met5]enkephalin; this was mimicked by DAMGO and blocked by CTOP.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, D-Penicillamine (2,5)-; Enkephalin, Methionine; Enkephalins; Glutamates; Glutamic Acid; Gyrus Cinguli; Naloxone; Narcotic Antagonists; Narcotics; Neurons; Neurotransmitter Agents; Rats; Receptors, Opioid; Somatostatin; Synapses; Synaptic Transmission

The roles of mu, delta and kappa opioid receptor subtypes in spinal morphine-induced antinociception were investigated. A C-fiber reflex elicited by electrical stimulation within the territory of the sural nerve was recorded from the ipsilateral biceps femoris muscle in anesthetized rats. Recruitment curves were built by varying the stimulus intensity from 0 to 7x threshold and temporal evolutions were studied by using a constant level of stimulus intensity (3x threshold). Intrathecal administration of naloxone, Cys2-Tyr3-Orn5-Pen7 amide (mu opioid receptor antagonist) and nor-binaltorphimine (nor-BNI, a kappa opioid receptor antagonist) completely antagonized the depression of the C-fiber reflex induced by 4 nmol of intrathecal morphine, whereas the antagonistic effect of naltrindole (a delta receptor antagonist) was limited, with a ceiling effect of 56%. The AD50 were 12 pmol and 1, 4.3 and 39 nmol for Cys2-Tyr3-Orn5-Pen7 amide, naloxone, nor-BNI and naltrindole, respectively. When injected alone, only naltrindole induced a short-duration depressive effect. Intrathecal administration of DAGO resulted in a depressive effect on the C-fiber reflex in a dose-dependent manner; for a stimulus intensity of 3x threshold, the ED50 was 9 pmol. DAGO was found to be 60 times more potent than morphine. Interestingly, nor-BNI, at doses which reversed the blockade of the C-fiber reflex by morphine, also reversed the effects of an equipotent dose of DAGO, which suggested an action on a mu receptor subtype.

Topics: Animals; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalins; Injections, Spinal; Morphine; Naltrexone; Narcotic Antagonists; Nerve Fibers; Rats; Rats, Sprague-Dawley; Receptors, Opioid, delta; Receptors, Opioid, kappa; Receptors, Opioid, mu; Reflex; Somatostatin

The contribution of high-frequency synaptic activity to the induction of long-term potentiation (LTP) in the opioid peptide-containing mossy fiber projection was investigated in vivo in anesthetized rats. Because high-frequency mossy fiber activity is essential for both the release of opioid peptides and the induction of mossy fiber LTP, we investigated whether the activation of opioid receptors underlies the requirement of sustained high-frequency mossy fiber activity for LTP induction. Mossy fiber responses were found to have a distinct threshold for the number of 100 Hz pulses necessary to induce LTP, with bursts of 25-30 pulses being the minimum for LTP induction. Application of 1 nmol of the mu-opioid receptor agonist DAMGO to the CA3 region potentiated mossy fiber responses, but, unlike for mossy fiber LTP, this potentiation could be reversed by mu-opioid receptor antagonist CTOP. Stimulation of the mossy fibers with either a single burst of 15 pulses at 100 Hz or application of 100 pmol of DAMGO was ineffective in potentiating mossy fiber responses. However, delivery of a 15 pulse burst 10 min following DAMGO application was effective in potentiating mossy fiber responses. This potentiation was not reversed by CTOP and it occluded stimulation-induced LTP, suggesting that brief bursts delivered in the presence of DAMGO had induced mossy fiber LTP. The release of opioid peptides and the resulting activation of mu-opioid receptors is suggested as one factor that underlies the requirement of sustained high-frequency stimulation for the induction of mossy fiber LTP.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Electric Stimulation; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalins; Hippocampus; Long-Term Potentiation; Male; Nerve Fibers; Rats; Rats, Sprague-Dawley; Receptors, Opioid; Somatostatin

A new class of models to describe antagonistic drug interactions are presented. They are semiparametric in that they use nonparametric functions (splines) but are forced to obey certain constraints corresponding to reasonable assumptions. We propose the models primarily for exploratory data analysis, but they may also be definitive models for such purposes as predicting future responses. Certain problems that arise in semiparametric modeling, such as model selection, are addressed so that we can propose a relatively automatic and objective approach to model determination. We demonstrate the applicability of the class of models we propose to two real data set examples involving pain relief response to opioid agonists/antagonists. The results suggest that the semiparametric approach is particularly useful when unusual shapes link dose to response.

Topics: Animals; Binding, Competitive; Drug Antagonism; Drug Interactions; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalins; Injections, Intraventricular; Models, Biological; Naltrexone; Narcotic Antagonists; Rats; Receptors, Opioid, delta; Receptors, Opioid, mu; Somatostatin

The mossy fiber-CA3 synapse displays an N-methyl-D-aspartate-receptor-independent mu-opioid-receptor-dependent form of long-term potentiation (LTP) that is thought not to display cooperativity or associativity with coactive afferents. However, because mossy fiber LTP requires repetitive synaptic activity for its induction, we reevaluated cooperativity and associativity at this synapse by using trains of mossy fiber stimulation. Moderate-, but not low-, intensity trains induced mossy fiber LTP, indicating cooperativity. Low-intensity mossy fiber trains that were normally ineffective in inducing LTP could induce mossy fiber LTP when delivered in conjunction with trains delivered to commissural-CA3 afferents. Associative mossy fiber LTP also could be induced with single mossy fiber pulses when delivered with commissural trains in the presence of a mu-opioid-receptor agonist. Our findings suggest a frequency-dependent variation of Hebbian associative LTP induction that is regulated by the release of endogenous opioid peptides.

Topics: Afferent Pathways; Animals; Electric Stimulation; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalins; Hippocampus; Long-Term Potentiation; Male; Nerve Fibers; Piperazines; Pyramidal Tracts; Rats; Rats, Sprague-Dawley; Receptors, N-Methyl-D-Aspartate; Receptors, Opioid, mu; Somatostatin; Synapses; Synaptic Transmission; Time Factors

The regulation of adenylyl cyclase by opioid receptor types was characterized in the rat nucleus accumbens, a brain region that is involved in the reinforcing effects of drugs of abuse, and in the caudate putamen, a region not implicated in drug reinforcement. Both mu and delta opioid ligands inhibited adenylyl cyclase activity in the nucleus accumbens and in the caudate putamen of rat, whereas the kappa agonist, U69,593 (5 alpha, 7 alpha, 8 alpha)-(+)-N-methyl-N-[7-(pyrrolidinyl)-1-oxaspiro [4,5]dec-8-yl]-benzeneacetamide, was ineffective. The mu agonists, DAMGO and Tyr-D-Arg-Phe-Sar, were more potent inhibitors of the enzyme in caudate putamen than in nucleus accumbens. The delta-selective agonists, DSLET and [D-Ala2]-deltorphin II more potently inhibited adenylyl cyclase in nucleus accumbens than in caudate putamen. Inhibition of the enzyme by DAMGO and Tyr-D-Arg-Phe-Sar was antagonized by the mu-selective competitive antagonist, CTOP D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2, and the noncompetitive mu antagonists, beta-funaltrexamine and naloxonazine. Inhibition of adenylyl cyclase activity by the delta-selective ligands, DPDPE, DSLET and [D-Ala2]-deltorphin II was unaffected by these antagonists. Conversely, the delta-selective antagonists, ICI 174,864 N-allyl2-Tyr-(alpha-aminisobutyric acid)2-Phe-Leu-OH and naltrindole, blocked the effects of the delta but not the mu opioid ligands. Adenylyl cyclase activity in nucleus accumbens and in caudate putamen is subject to regulation by both mu and delta opioid receptors.

Topics: Adenylyl Cyclases; Amino Acid Sequence; Animals; Benzeneacetamides; Caudate Nucleus; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, D-Penicillamine (2,5)-; Enkephalin, Leucine; Enkephalins; Male; Molecular Sequence Data; Naloxone; Naltrexone; Nucleus Accumbens; Oligopeptides; Putamen; Pyrrolidines; Rats; Rats, Sprague-Dawley; Receptors, Opioid, delta; Receptors, Opioid, mu; Signal Transduction; Somatostatin

In membranes from SH-SY5Y human neuroblastoma cells differentiated with retinoic acid, the mu-selective agonist Tyr-D-Ala-Gly-N-Me-Phe-Gly-ol (DAMGO) inhibited cAMP formation with an IC50 of 26 nM. Two separate antibodies raised against distinct regions of the Go alpha sequence attenuated the effect of DAMGO by 50-60%, whereas antibodies to Gi alpha 1,2 or Gi alpha 3 reduced the mu-opioid signal insignificantly or to a lesser extent. In contrast, inhibition of adenylyl cyclase by the delta-opioid agonist Tyr-D-Pen-Gly-Phe-D-Pen-OH (DPDPE; Pen = penicillamine) was very sensitive to the Gi alpha 1,2 antibody. In membranes from rat brain striatum, coupling of the mu opioid receptor to adenylyl cyclase was also maximally blocked by antibodies to Go alpha. After long-term treatment of the cells with DAMGO, the content of Go alpha was reduced by 26%, whereas the levels of Gi alpha 1,2, Gi alpha 3, and Gs alpha were unaltered. Addition of Go, purified from bovine brain, to membranes from pertussis toxin-treated SH-SY5Y cells restored the inhibition of adenylyl cyclase by DAMGO to 70% of that in toxin-untreated cells. To comparably restore the effect of DPDPE, much higher concentrations of Go were required. By demonstrating mediation of cAMP-dependent signal transduction by Go, these results describe (i) an additional role for this G protein present at a high concentration in brain, (ii) preferential, although not exclusive, interaction of mu and delta opioid receptors with different G protein subtypes in coupling to adenylyl cyclase, and (iii) reduced levels of Go following chronic opioid treatment of SH-SY5Y cells with mu opioids.

Topics: Adenylate Cyclase Toxin; Adenylyl Cyclases; Analgesics; Animals; Antibodies; Cell Membrane; Clone Cells; Corpus Striatum; Cyclic AMP; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, D-Penicillamine (2,5)-; Enkephalins; GTP-Binding Proteins; Humans; Kinetics; Membrane Proteins; Neuroblastoma; Pertussis Toxin; Rats; Receptors, Opioid, mu; Somatostatin; Tumor Cells, Cultured; Virulence Factors, Bordetella

Intracellular recordings were made from neurons in the lateral nucleus of the amygdala, in a slice of rat brain that was superfused in vitro. [Met5]enkephalin (3-30 microM) and the mu receptor selective agonist DAMGO (Tyr-D-Ala-Gly-MePhe-Gly-ol; 0.3-3 microM) hyperpolarized about 50% of cells; this was blocked by naloxone and by the mu receptor antagonist CTOP (D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2). The pA2s for naloxone and CTOP were 8.3 and 7.7, respectively. DPDPE (Tyr-D-Pen-Gly-Phe-D-Pen: delta receptor selective) and U50488 (trans-(+-)-3,4-dichloro-N-methyl-[2-(1-pyrrolidinyl)cyclohexyl] benzeneacetamide methane sulfonate; kappa receptor selective) had no effect. Synaptic potentials mediated by gamma-aminobutyric acid (GABA) acting at GABAA receptors were elicited by focal stimulation of the slice in a combination of 6-cyano-2,3-dihydroxy-7-nitroquinoxaline (10 microM) and 4-aminophosphonovaleric acid (30 microM). They were inhibited by up to 60% by DAMGO and by DPDPE. The action of DAMGO was blocked by CTOP but not by the delta-selective antagonist ICI174864 (N,N-bisallyl-Tyr-Aib-Aib-Phe-Leu-OH, Aib = aminoisobutyrate). The action of DPDPE was blocked by ICI174864 but not by CTOP. Depolarizations elicited by addition of GABA to the superfusing solution were not affected by opioids. It is concluded that activation of mu opioid receptors hyperpolarizes about 50% of lateral amygdala neurons. Activation of either mu or delta receptors also inhibits presynaptically the release of GABA.

Topics: 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; Amino Acid Sequence; Amino Acids; Amygdala; Analgesics; Animals; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, D-Penicillamine (2,5)-; Enkephalin, Leucine; Enkephalin, Methionine; Enkephalins; GABA-A Receptor Antagonists; In Vitro Techniques; Molecular Sequence Data; Naloxone; Narcotic Antagonists; Narcotics; Neurons; Pyrrolidines; Rats; Somatostatin; Synapses

The present voltage-clamp data from guinea pig colonic mucosa indicate that mu opioid receptors both enhance and inhibit net chloride secretion stimulated by PGE1 plus theophylline. Actually, enhancement by the mu opioid agonist DAGO (0.2 nmol/l) declines and finally turns into inhibition with increasing net chloride secretion observed in control preparations taken from the same animals. Moreover, inhibition by the mu opioid antagonist CTOP-NH2 (100 nmol/l) increases with increasing inhibition (or decreasing enhancement) by the mu opioid agonist DAGO. Thus, the mu opioid agonist DAGO and the mu opioid antagonist CTOP-NH2 display opposite effects on net chloride secretion only to a limited degree but otherwise show parallel, either stimulatory or inhibitory, effects. To explain this complex function of mu opioid receptors in the control of net chloride secretion, a working hypothesis is being proposed.

Topics: Alprostadil; Amiloride; Amino Acid Sequence; Animals; Chlorides; Colon; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalins; Guinea Pigs; In Vitro Techniques; Intestinal Mucosa; Male; Molecular Sequence Data; Receptors, Opioid, mu; Somatostatin; Theophylline

The mesolimbic dopaminergic system has been implicated in mediating the motivational effects of opioids and other drugs of abuse. The site of action of opioids within this system and the role of endogenous opioid peptides in modulating dopamine activity therein remain unknown. Employing the technique of in vivo microdialysis and the administration of highly selective opioid ligands, the present study demonstrates the existence of tonically active and functionally opposing mu and kappa opioid systems that regulate dopamine release in the nucleus accumbens, the major terminal area of A10 dopaminergic neurons. Thus, stimulation of mu-type receptors in the ventral tegmental area, the site of origin of A10 dopaminergic neurons, increases dopamine release whereas the selective blockade of this opioid receptor type results in a significant decrease in basal dopamine release. In contrast, stimulation of kappa-type receptors within the nucleus accumbens decreases dopamine release whereas their selective blockade markedly increases basal dopamine release. These data show that tonic activation of mu and kappa receptors is required for the maintenance of basal dopamine release in the nucleus accumbens. In view of the postulated role of the mesolimbic system in the mediation of drug-induced alterations in mood and affect, such findings may have implications for the treatment of opiate dependence and affective disorders.

Topics: Analgesics; Animals; Benzeneacetamides; Dopamine; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalins; Infusions, Parenteral; Limbic System; Male; Microinjections; Models, Neurological; Naltrexone; Nucleus Accumbens; Pyrrolidines; Rats; Rats, Inbred Strains; Somatostatin; Tegmentum Mesencephali

Bilateral injection of naloxone (3.0-30.0 nmol) into the substantia nigra of morphine-dependent rats produced a withdrawal syndrome consisting of wet-dog shakes, teeth chattering, irritability to touch, diarrhea and hypothermia. Intense wet-dog shakes and grooming were observed after intranigral injection of the mu selective antagonist D-Phe-Cys-Try-D-Trp-Orn-Thr-Pen-Thr-NH2 (CTOP, 3.0-30.0 nmol) in morphine-dependent animals. Body temperature after 30.0 nmol CTOP was significantly increased. A significant positive correlation between body temperature and wet-dog shakes was observed in morphine-dependent animals that received CTOP. Intranigral injection of beta-funaltrexamine (beta-FNA, 10.0 nmol), an irreversible mu antagonist, produced no signs of withdrawal in morphine-dependent animals. However, intranigral injection of beta-FNA (1.0-3.0 nmol) suppressed the antinociceptive effect of the mu-selective agonist, D-Ala2,N-Me-Phe4,Gly5-ol-enkephalin (DAGO, 1.0 nmol). The withdrawal syndrome produced by CTOP (10.0 nmol) was not suppressed by the administration of U50,488H (10.0 nmol), a kappa agonist, suggesting that the absence of an effect of beta-FNA was not due to its kappa agonist activity. Neither the delta-selective antagonist, naltrindole (NTI, 10.0 nmol) nor the kappa-selective antagonist, nor-binaltorphimine (nor-BNI, 10.0 nmol) produced withdrawal. Only wet-dog shakes were observed when CTOP, NTI and nor-BNI (5 nmol each) were administered together into the nigra. These studies suggest an involvement of mu receptors in the nigra in the wet-dog shakes and thermoregulatory dysfunction that occur during withdrawal of morphine. However, the subtypes of opioid receptors in the nigra, that mediate the other signs of morphine withdrawal remain obscure.

Topics: Amino Acid Sequence; Analgesics; Animals; Behavior, Animal; Body Temperature; Brain; Dose-Response Relationship, Drug; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalins; Male; Molecular Sequence Data; Morphine; Morphine Dependence; Naloxone; Rats; Rats, Sprague-Dawley; Receptors, Opioid; Somatostatin; Stereotaxic Techniques; Substance Withdrawal Syndrome; Substantia Nigra

The effect of intracerebroventricular administration of a selective mu- (CTOP) or delta- (ICI 174,864) opioid receptor antagonist on the antinociceptive effects produced by intrathecal administration of selective mu- (DAMGO), delta- (DPDPE) and kappa- (U50-488H) opioid receptor agonists was evaluated using the Randall-Selitto paw-withdrawal test, in the rat. While the intracerebroventricular administration of CTOP or ICI 174,864, alone, had no effect on nociceptive thresholds, intracerebroventricular administration of CTOP and ICI 174,864 produced marked antagonism of the antinociceptive effects of intrathecal DAMGO. The antinociceptive effects of intrathecal administration of DPDPE or U50,488H were not antagonized by intracerebroventricular administration of CTOP or ICI 174,864. These data suggest that, in the rat, along with the established descending antinociceptive pathways, there is an ascending antinociceptive control mechanism projecting from the spinal cord to the brainstem. The ascending antinociceptive control involves mu- and delta-opioid agonism at supraspinal sites and appears to be mediated selectively by mu-, but not by delta- or kappa-opioid agonism at the spinal level.

Topics: 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; Analgesics, Opioid; Animals; Dose-Response Relationship, Drug; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, D-Penicillamine (2,5)-; Enkephalin, Leucine; Enkephalins; Injections, Intraventricular; Injections, Spinal; Male; Narcotic Antagonists; Pain Threshold; Pyrrolidines; Rats; Rats, Sprague-Dawley; Somatostatin

The effects on one-way active avoidance conditioning of pre-training, systemic administration of the selective mu-receptor agonist [D-Ala2,N-Me-Phe4, Gly-ol]enkephalin (DAGO), and the selective mu-receptor antagonist (D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2 (CTOP), were determined in Swiss-Webster mice. A low dose of DAGO (0.92 micrograms/kg) moderately enhanced avoidance acquisition, whereas a 100 micrograms/kg dose of CTOP more dramatically impaired acquisition. However, the avoidance-enhancing dose of DAGO significantly increased locomotor activity as measured in a separate group of mice in the avoidance chamber, and the avoidance-impairing dose of CTOP significantly decreased activity. Under these same training conditions, earlier studies (Schulteis et al. 1988; Schulteis and Martinez 1990) demonstrated that enkephalins impaired avoidance learning, and selective delta-receptor antagonists such as ICI 174,864 enhanced learning; in contrast to the present study, both of these effects were dissociated from performance effects such as alterations in locomotor activity. Taken together, the results suggested that the effects of enkephalins were mediated by the delta-, but not mu-, class of opioid receptor.

Topics: Amino Acid Sequence; Analgesics; Animals; Avoidance Learning; Electroshock; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalins; Male; Mice; Molecular Sequence Data; Motor Activity; Receptors, Opioid, delta; Receptors, Opioid, mu; Somatostatin

Delta opioid binding sites were assayed using [3H][D-ala2,D-leu5]enkephalin and rat brain membranes depleted of mu binding sites with the site-directed acylating agent, 2-(p-ethoxybenzyl)-1-diethylaminoethyl-5-isothiocyanatobenzimid a zole-HCI. [D-Pen2,D-Pen5]enkephalin (DPDPE), [D-Pen2,L-Pen5]enkephalin, [D-Ala2]deltorphin-I and [D-Ala2]deltorphin-II inhibition curves were characterized by slope factors (Hill coefficients) less than 1. The low slope factor of DPDPE persisted in the presence of 50 microM 5'-guanylyimidodiphosphate in the assay Quantitative analysis of [D-ala2,D-leu5]enkephalin, DPDPE and [D-Ala2]deltorphin-I binding surfaces resolved two binding sites. Whereas [D-ala2,D-leu5]enkephalin had equal affinity for both sites, DPDPE and [D-Ala2]deltorphin-I had high affinity for the high capacity binding site, and low affinity for the low capacity binding site. These data support pharmacological studies demonstrating delta receptor subtypes which mediate antinociception.

Topics: Amino Acid Sequence; Animals; Binding Sites; Brain; Cell Membrane; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, D-Penicillamine (2,5)-; Enkephalin, Leucine-2-Alanine; Enkephalins; Kinetics; Molecular Sequence Data; Oligopeptides; Radioligand Assay; Rats; Receptors, Opioid; Receptors, Opioid, delta; Somatostatin

Our previous work suggests that opioid peptides modulate sensory nerves in the perfused rat mesentery. Therefore we tested the hypothesis that opioids are involved in the ongoing regulation of sensory nerve activity using selective opioid receptor antagonists. In the presence of guanethidine and methoxamine, transmural nerve stimulation caused a vasodilator response which was potentiated significantly by naloxone (3 x 10(-7) M). However, naloxone did not affect vasodilator responses to exogenous calcitonin gene related peptide. IC1 174.864 (3 x 10(-7) M), a selective delta receptor antagonist, had no effect on vasodilator responses to transmural nerve stimulation. In contrast CTOP (D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Phe-Thr-NH2) (3 x 10(-7) M), a selective mu receptor antagonist, significantly inhibited vasodilator responses to transmural nerve stimulation, effects which were abolished by naloxone treatment. In preparations pretreated with beta-FNA (beta-funaltrexamine HCl), an irreversible mu receptor antagonist, naloxone no longer potentiated vasodilator responses to transmural nerve stimulation. These results suggest that potentiation of vasodilator responses to transmural nerve stimulation by naloxone may be due to blockade of mu receptors, resulting in a reduced inhibitory modulation by endogenous opioids. These findings support the contention that prejunctional opioid receptors on sensory nerves may play a role in modulating activity of the cardiovascular system.

Topics: Animals; Calcitonin Gene-Related Peptide; Electric Stimulation; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, Leucine; Enkephalins; Guanethidine; In Vitro Techniques; Male; Mesenteric Arteries; Methoxamine; Muscle, Smooth, Vascular; Naloxone; Naltrexone; Narcotic Antagonists; Neurons, Afferent; Perfusion; Rats; Rats, Inbred Strains; Somatostatin; Vasodilation

The influence of four opioid antagonists on short circuit current (Isc), transepithelial potential difference (Pdo) and tissue conductance (Gt) in the guinea pig colonic mucosa was investigated in vitro under both basal and PGE1 plus theophylline-stimulated conditions. The experiments aimed at identifying the opioid receptor type(s) endogenously activated to control chloride secretion. Under blockade of sodium-dependent Isc by amiloride (100 mumol/l), net anion secretion was regarded to equal the lumen-negative shift in Isc upon addition of 1 mumol/l PGE1 plus 100 mumol/l theophylline. It was significantly elevated by 100 nmol/l of the kappa-selective antagonist nor-binaltorphimine (nor-BNI). This augmenting effect was totally abolished in amiloride-free buffer or by omission of chloride. 1 mumol/l TTX completely prevented the effect of both PGE1 plus theophylline and nor-BNI. Both the kappa agonist U 69593 (10 nmol/l) and the calcium channel agonist Bay K 8644 (1 mumol/l) significantly depressed net anion secretion stimulated by PGE1 plus theophylline. Nor-BNI at 10 nmol/l prevented the suppressive effect of both Bay K 8644 and U 69593. This suggests release of endogenous opioids by the calcium channel agonist Bay K 8644 and competition between the kappa agonist U 69593 and the kappa antagonist nor-BNI. In contrast to the kappa antagonist nor-BNI, the mu antagonist CTOP-NH2 at 100 nmol/l significantly impaired, while the mu-selective agonist DAGO at 0.2 nmol/l augmented, net anion secretion stimulated by PGE1 plus theophylline. The effect of CTOP-NH2 was abolished in chloride-free buffer.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: 3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester; Alprostadil; Amiloride; Amino Acid Sequence; Analgesics; Animals; Benzeneacetamides; Chlorides; Endorphins; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalins; Gluconates; Guinea Pigs; Intestinal Mucosa; Male; Membrane Potentials; Molecular Sequence Data; Naltrexone; Narcotic Antagonists; Pyrrolidines; Receptors, Opioid; Receptors, Opioid, delta; Receptors, Opioid, kappa; Receptors, Opioid, mu; Somatostatin; Tetrodotoxin; Theophylline

Topics: Animals; Binding, Competitive; Brain; Diprenorphine; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, D-Penicillamine (2,5)-; Enkephalins; Guinea Pigs; In Vitro Techniques; Indoles; Morphinans; Naltrexone; Receptors, Opioid; Receptors, Opioid, delta; Receptors, Opioid, mu; Somatostatin

An unbiased place preference conditioning procedure was used to identify the central opioid receptor types through which the endogenous opioid peptide, beta-endorphin, acts to exert its reinforcing effects in rats in vivo. The intracerebroventricular administration of beta-endorphin, and selective mu (DAGO) or delta (DPDPE) opioid receptor agonists produced marked preferences for the drug-associated place. Intracerebroventricular pretreatment with the selective mu antagonist, CTOP, eliminated the place preference produced by DAGO but not that produced by DPDPE. Pretreatment with the selective delta antagonist, ICI 174,864, abolished the place preference induced by DPDPE. It did not modify the effect of DAGO. In contrast, pretreatment with either ICI 174,864 or CTOP abolished the effects of beta-endorphin. These data demonstrate that both mu and delta receptors are involved in mediating the reinforcing effect of beta-endorphin and indicate that the activation of both receptor types is required for the expression of the motivational effects of beta-endorphin. Further they suggest that beta-endorphin produces its motivational effects via an interaction with an opioid receptor complex composed of both mu and delta receptors.

Topics: Amino Acid Sequence; Animals; beta-Endorphin; Conditioning, Operant; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, D-Penicillamine (2,5)-; Enkephalin, Leucine; Enkephalins; Male; Molecular Sequence Data; Narcotic Antagonists; Rats; Rats, Inbred Strains; Receptors, Opioid; Receptors, Opioid, delta; Receptors, Opioid, mu; Reinforcement, Psychology; Somatostatin

The effects of intracerebroventricular (i.c.v.) administration of D-Phe-Cys-Tyr-D-Try-Orn-Thr-Pen-Thr-NH2 (CTOP), a selective mu-opioid receptor antagonist, (Allyl)2-Tyr-Aib-Aib-Phe-Leu-OH (ICI 174864) and (N,N-Bisallyl-Tyr-Gly-Gly-psi-(CH2S)-Phe-Leu-OH (ICI 154129), selective delta-opioid receptor antagonists on blocking analgesia induced by beta-endorphin, morphine, D-Ala2-NMePhe4-Gly-ol-enkephalin (DAMGO), D-Ala2-D-Leu5-enkephalin (DADLE) and D-Pen2-enkephalin (DPDPE) administered i.c.v. were studied in male ICR mice. The analgesia was assessed by the tail-flick and paw-licking (hot-plate) tests. The potencies of opioid agonists injected i.c.v. for producing analgesia were DAMGO greater than DADLE greater than beta-endorphin greater than morphine greater than DPDPE. Intracerebroventricular administration of CTOP (0.05 micrograms) selectively antagonized inhibition of the tail-flick and paw-licking response induced by morphine, DAMGO or DADLE but not beta-endorphin or DPDPE. ICI 174864 (5 micrograms) and ICI 154129 (5 micrograms) injected i.c.v. selectively antagonized analgesia induced by DPDPE or DADLE but not beta-endorphin, morphine or DAMGO injected i.c.v. These results indicate that analgesia induced by morphine and DAMGO is mediated by the stimulation of mu-opioid receptors while analgesia induced by DPDPE is mediated by the stimulation of delta-opioid receptors. DADLE-induced analgesia is mediated by the stimulation of both mu- and delta-opioid receptors. Analgesia induced by beta-endorphin is mediated by neither mu- nor delta-opioid receptors.

Topics: Analgesia; Animals; beta-Endorphin; Endorphins; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, D-Penicillamine (2,5)-; Enkephalin, Leucine; Enkephalin, Leucine-2-Alanine; Enkephalins; Injections, Intraventricular; Male; Mice; Mice, Inbred ICR; Morphine; Narcotic Antagonists; Reaction Time; Receptors, Opioid; Somatostatin

In the present study we used in vivo microdialysis to examine the influence of beta-endorphin on dopamine (DA) release in the nucleus accumbens of anesthetized rats and to identify the opioid receptor types mediating its effects. Microdialysis probes were inserted into the nucleus accumbens and perfusates were analysed for DA and its metabolites, dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), using a reversed phase HPLC system with electrochemical detection for separation and quantification. Intracerebroventricular (i.c.v.) administration of beta-endorphin resulted in a dose-dependent increase in DA and its metabolites. Pretreatment with the selective delta-antagonist ICI 174,864 significantly attenuated the beta-endorphin-induced increase in DA release and metabolism whereas pretreatment with the selective mu-antagonist CTOP resulted abolition of the beta-endorphin effect. These data demonstrate that the blockade of either mu- or delta-opioid receptors is sufficient to antagonize the stimulatory effects of beta-endorphin on DA release and metabolism. As such, these findings suggest that the concomitant activation of both mu- and delta-receptors underlies the effects of beta-endorphin on DA release in the nucleus accumbens.

Topics: Analgesics; Animals; beta-Endorphin; Biogenic Monoamines; Brain Chemistry; Chromatography, High Pressure Liquid; Dialysis; Dopamine; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, D-Penicillamine (2,5)-; Enkephalin, Leucine; Enkephalins; Injections, Intraventricular; Male; Narcotic Antagonists; Nucleus Accumbens; Rats; Rats, Inbred Strains; Receptors, Opioid; Somatostatin