u-50488 and deltorphin

The effect exerted by two gamma-endorphin derivatives (DTgammaE and DEgammaE) was investigated on morphine-induced inhibition on the electrically induced contractions of guinea pig ileum in vitro. Morphine (1x10(-8)-5x10 (-8)-1x10 (-7) M) dose dependently and significantly reduced the E.C. of guinea pig ileum, IC50=6.5x10(-8) M (Confidence limits: 3.7x10 (-8)-9.1x10 (-8)). DTgammaE and DEgammaE per se (1x10 (-6)-5x10 (-6)-1x10 (-5) M) did not modify significantly the E.C. of guinea pig ileum. Furthermore, DTgammaE or DEgammaE injection 10-30-60 min before morphine, did not affect the inhibitory effect of morphine on the E.C. of guinea pig ileum. By contrast, ilea from guinea-pigs treated for 4 days with DTgammaE or DEgammaE (1 mg/Kg/i.p.) were less sensitive to the inhibitory effect of morphine, IC50=8.3x10 (-7) M (Confidence limits: 1.4x10(-6)-3.5x10(-7)) for DTgammaE and IC50=7.7x10(-7) M (Confidence limits: 2.7x10(-6)-8.7x10(-7 )) for DEgammaE . The effect exerted by two beta-endorphin fragments (DTgammaE and DEgammaE) was investigated on the acute opiate withdrawal induced by micro, kappa and delta receptor agonists in vitro. After a exposure in vitro for 4 min to morphine (less selective micro agonist), DAGO (highly selective micro agonist), U50-488H (highly selective kappa agonist) and beta-endorphin (selective micro- delta agonist), a strong contracture of isolated guinea pig ileum was observed after the addition of naloxone. This effect was also observed when isolated rabbit jejunum was pretreated with deltorphin (highly selective delta agonist). DTgammaE or DEgammaE injection before or after treatment with morphine, DAGO, U50-488H, beta-endorphin or deltorphin was able of both preventing and reversing the naloxone-induced contracture after exposure to the opioid agonists in a concentration-dependent fashion. Our results indicate that both DTgammaE or DEgammaE are able to reduce significantly opiate withdrawal in vitro, suggesting an important functional interaction beween beta-endorphin fragments and opioid withdrawal at both micro, kappa and delta receptor level. Our results indicate that chronic treatment of guinea pigs with DTgammaE or DEgammaE induces a significant reduction of the inhibitory effect of morphine on the E.C. of guinea-pig ileum thus confirming an important functional interaction between gamma-endorphin derivatives and opioid system.

Topics: 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; Acetylcholine; Animals; beta-Endorphin; Electric Stimulation; Endorphins; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Guinea Pigs; Ileum; In Vitro Techniques; Jejunum; Male; Morphine; Muscle Contraction; Muscle, Smooth; Naloxone; Oligopeptides; Peptide Fragments; Rabbits; Substance Withdrawal Syndrome

Zymosan-induced peritoneal inflammation was inhibited by morphine co-injection in mice and fish but not in anuran amphibians. In present experiments, an in vitro migration of mouse, goldfish, and frog leukocytes to L15 medium, control serum (S) or zymosan-activated serum (ZAS) was recorded following cell preincubation with L15 or with agonists of mu, delta, or kappa opioid receptors (morphine, deltorphine, or U-50,488H, respectively). In all species, migration of control leukocytes was in the order ZAS > S > L15. Pretreatment with morphine or deltorphine (but not with U-50,488H) enhanced leukocyte migration to L15 and S in each species, while it inhibited migration of mouse and fish (but not frog) leukocytes to ZAS, phenomena reversed by specific antagonists of mu and delta opioid receptors (CTOP or naltrindole, respectively). It seems that final effects of opioids on cell migration are dependent on a species-specific balance between up- and down-regulation of leukocyte migration resulted from interplay between receptors for opioids and chemotactic factors.

Topics: 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; Analgesics, Non-Narcotic; Analgesics, Opioid; Animals; Chemotaxis, Leukocyte; Culture Media, Serum-Free; Enzyme Precursors; Goldfish; Mice; Morphine; Oligopeptides; Organic Chemicals; Rana esculenta; Receptors, Opioid

The peptide nociceptin/orphanin FQ (N/OFQ) and its receptor ORL-1, also designated opioid receptor 4 (OP(4)) are involved in the modulation of nociception. Using OP(4)-knockout mice, we have studied their response following opioid receptor stimulation and under neuropathic conditions.In vas deferens from wild-type and OP(4)-knockout mice, DAMGO (mu/OP(3) agonist), deltorphine II (delta/OP(1) agonist) and (-)-U-50488 (kappa/OP(2) agonist) induced similar concentration-dependent inhibition of electrically-evoked contractions. Naloxone and naltrindole (delta/OP(1) antagonists) shifted the curves of DAMGO (pA(2)=8.6) and deltorphine II (pA(2)=10.2) to the right, in each group. In the hot-plate assay, N/OFQ (10 nmol per mouse, i.t.) increased baseline latencies two-fold in wild-type mice while morphine (10mg/kg, s.c.), deltorphine II (10 nmol per mouse, i.c.v.) and dynorphin A (20 nmol per mouse, i.c.v.) increased hot-plate latencies by about four- to five-fold with no difference observed between wild-type and knockout mice. Furthermore, no change was evident in the development of the neuropathic condition due to chronic constriction injury (CCI) of the sciatic nerve, after both thermal and mechanical stimulation. Altogether these results suggest that the presence of OP(4) receptor is not crucial for (1) the development of either acute or neuropathic nociceptive responses, and for (2) the regulation of full receptor-mediated responses to opioid agonists, even though compensatory mechanisms could not be excluded.

Topics: 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; Analgesics, Non-Narcotic; Analgesics, Opioid; Animals; Dose-Response Relationship, Drug; Dynorphins; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Female; Male; Mice; Mice, Knockout; Morphine; Naloxone; Naltrexone; Narcotic Antagonists; Nociceptin; Nociceptin Receptor; Oligopeptides; Opioid Peptides; Pain; Receptors, Opioid; Time Factors; Vas Deferens

Projection neurons of the striatum release opioid peptides in addition to GABA. Our previous studies showed that the opioid peptide dynorphin regulates that subtype of projection neurons which sends axons to the substantia nigra/entopeduncular nucleus, as indicated by an inhibitory action of dynorphin/agonists on D1 dopamine receptor-mediated immediate-early gene induction in these neurons. The other subtype of striatal projection neurons projects to the globus pallidus and contains the opioid peptide enkephalin. Here, we investigated whether enkephalin regulates the function of striatopallidal neurons, by analysing opioid effects on immediate-early gene induction by D2 dopamine receptor blockade that occurs in these neurons. Thus, the effects of systemic and intrastriatal administration of various opioid receptor agonists and antagonists on immediate-early gene expression (c-fos, zif 268) induced by the D2 receptor antagonist eticlopride were examined with in situ hybridization histochemistry. Intrastriatal infusion of enkephalin (delta and mu), but not dynorphin (kappa), receptor agonists suppressed immediate-early gene induction by eticlopride in a dose-dependent manner. This suppression was blocked by the opioid receptor antagonist naloxone, confirming the involvement of opioid receptors. Repeated treatment with D2 receptor antagonists produces increased enkephalin expression and diminished immediate-early gene inducibility in striatopallidal neurons, as well as behavioral effects that are attenuated compared to those of acute treatment (e.g., reduced akinesia). Naloxone reversed such behavioral recovery (i.e. reinstated akinesia), but did not significantly affect suppressed immediate-early gene induction. Our results indicate that enkephalin acts, via mu and delta receptors in the striatum, to inhibit acute effects of D2 receptor blockade in striatopallidal neurons. Moreover, the present findings suggest that increased enkephalin expression after repeated D2 receptor antagonist treatment is an adaptive response that counteracts functional consequences of D2 receptor blockade, but is not involved in suppressed immediate-early gene induction. Together with our earlier findings of the role of dynorphin, these results indicate that opioid peptides in the striatum serve as negative feedback systems to regulate the striatal output pathways in which they are expressed.

Topics: 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; Analgesics; Animals; Base Sequence; Corpus Striatum; DNA Primers; DNA-Binding Proteins; Dopamine Antagonists; Dopamine D2 Receptor Antagonists; Early Growth Response Protein 1; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, Leucine-2-Alanine; Enkephalins; Gene Expression Regulation; Genes, fos; Genes, Immediate-Early; Immediate-Early Proteins; In Situ Hybridization; Male; Molecular Sequence Data; Naloxone; Neurons; Oligopeptides; Proto-Oncogene Proteins c-fos; Rats; Rats, Sprague-Dawley; Salicylamides; Transcription Factors; Transcriptional Activation

Most electrophysiological studies of opioids on hippocampal principal neurons have found indirect actions, usually through interneurons. However, our laboratory recently found reciprocal alteration of the voltage-dependent K(+) current, known as the M-current (I(M)), by kappa and delta opioid agonists in CA3 pyramidal neurons. Recent ultrastructural studies have revealed postsynaptic delta opiate receptors on dendrites and cell bodies of CA1 and CA3 hippocampal pyramidal neurons (HPNs). Reasoning that previous electrophysiological studies may have overlooked voltage-dependent postsynaptic effects of the opioids in CA1, we reevaluated their role in CA1 HPNs using the rat hippocampal slice preparation for intracellular current- and voltage-clamp recording. None of the delta and mu; receptor-selective opioids tested, including [D-Pen(2,5)]-enkephalin (DPDPE), [D-Ala(2)]-deltorphin II (deltorphin), [D-Ala(2), NMe-Phe(4), Gly-ol]-enkephalin (DAMGO), and [D-Ala(2), D-Leu(5)] enkephalin (DADLE), altered membrane properties such as I(M) or Ca(2+)-dependent spikes in CA1 HPNs. The nonopioid, Des-Tyr-dynorphin (D-T-dyn), also had no effect. By contrast, dynorphin A (1-17) markedly increased I(M) at low concentrations and caused an outward current at depolarized membrane potentials. The opioid antagonist naloxone and the kappa receptor antagonist nor-binaltorphimine (nBNI) blocked the I(M) effect. However, the kappa-selective agonists U69,593 and U50,488h did not significantly alter I(M) amplitudes when averaged over all cells tested, although occasional cells showed an I(M) increase with U50,488h. Our results suggest that dynorphin A postsynaptically modulates the excitability of CA1 HPNs through opiate receptors linked to voltage-dependent K(+) channels. These findings also provide pharmacological evidence for a functional kappa opiate receptor subtype in rat CA1 HPNs but leave unanswered questions on the role of delta receptors in CA1 HPNs.

Topics: 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; Animals; Benzeneacetamides; Dynorphins; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, D-Penicillamine (2,5)-; Enkephalin, Leucine-2-Alanine; Hippocampus; In Vitro Techniques; Oligopeptides; Peptide Fragments; Potassium Channels; Pyramidal Cells; Pyrrolidines; Rats; Receptors, Opioid; Receptors, Opioid, delta; Receptors, Opioid, kappa

Microinjection of [D-Ala2,MePhe4,Gly(ol)5]enkephalin (DAMGO) into either the periaqueductal gray (PAG) or the rostral ventral medulla (RVM) elicits analgesia in the tailflick assay in the rat. Co-administration of DAMGO into both regions together results in a profound synergistic interaction similar to that we previously reported with morphine. U50,488H and DPDPE are inactive when given into either region. [D-Ala2,Glu4]Deltorphin (deltorphin), on the other hand, elicits an analgesic response, although the maximal response is less than than mu agonists. Co-administration of DAMGO into one region with deltorphin in the other also results in a significant synergy. However, co-administration of DAMGO and deltorphin together in the same region gives only additive effects. These results confirm the existence of mu/mu synergy between the PAG and RVM. kappa 1 and delta 1 agents are inactive, but the delta 2 agonist deltorphin is active in both regions. Our results indicate the presence of mu/delta 2 synergy between the PAG and RVM which appears to involve interactions of pathways rather than receptor interactions at the cellular level.

Topics: 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; Amino Acid Sequence; Analgesics; Animals; Drug Synergism; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, D-Penicillamine (2,5)-; Enkephalins; Male; Medulla Oblongata; Molecular Sequence Data; Oligopeptides; Pain; Periaqueductal Gray; Pyrrolidines; Rats; Rats, Sprague-Dawley; Receptors, Opioid, delta; Receptors, Opioid, mu

In this work, we have characterized the opioid receptor expressed by the human neuroblastoma cell line SK-N-BE and compared its hydrodynamic behaviour with those of well known opioid receptors: mu-opioid receptor of rabbit cerebellum and delta-opioid receptor of the hybrid cell line NG 108-15. Human neuroblastoma cell line SK-N-BE expresses a substantial amount of opioid receptors (200-300 fmoles/mg of protein). Pharmacological characterization suggests an heterogenous population of receptors and the presence of two delta subtypes which are, at least partially, negatively coupled with adenylate cyclase via a Gi protein. These receptors exist under two different molecular forms and, in this respect, strikingly contrast with the archetypic delta receptors of NG 108-15 hybrid cell line which show only a high molecular weight form and appear more tightly coupled with the G protein. Hydrodynamic behaviour of SK-N-BE opioid receptors is reminiscent of the profile observed with the rabbit cerebellum mu-opioid receptor. This observation is consistent with the presence of two delta-opioid receptors subtypes, one of which exhibiting properties close to those of mu opioid receptors. Taken overall, our results suggest that different types and subtypes of opioid receptors, even if they are coupled to the same inhibitory G protein, are more or less tightly coupled with their transduction proteins and that closely related opioid receptors can form allosterically interacting complexes.

Topics: 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; Adenylyl Cyclases; Amino Acid Sequence; Animals; Binding, Competitive; Cerebellum; Colforsin; Diprenorphine; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, D-Penicillamine (2,5)-; Enkephalins; Etorphine; GTP-Binding Proteins; Guinea Pigs; Humans; Hybrid Cells; Molecular Sequence Data; Morphine; Naloxone; Neoplasm Proteins; Nerve Tissue Proteins; Neuroblastoma; Oligopeptides; Pyrrolidines; Rabbits; Receptors, Opioid; Receptors, Opioid, delta; Receptors, Opioid, mu; Tumor Cells, Cultured

1. Following a 5 min in vitro exposure to morphine (1.3 x 10(-7) M), U-50,488H (2.5 x 10(-8) M) and deltorphin (1.6 x 10(-8)-6.5 x 10(-9) M), the rabbit isolated jejunum exhibited a precipitated contracture after the addition of naloxone (2.75 x 10(-7) M). 2. The precipitated responses to U-50,488H and deltorphin but not to morphine were reproducible in the same tissue. 3. The precipitated contractures were blocked completely by tetrodotoxin (3 x 10(-7) M), partially by atropine (1.5 x 10(-7) M) and not affected by hexamethonium (1.4 x 10(-5) M). 4. Naloxone administration (2.75 x 10(-7) M) before the agonist prevented the development of the adaptive response to morphine and U-50,488H but not to deltorphin. 5. The selective antagonists norbinaltorphimine (2.7 x 10(-8)-2.7 x 10(-9) M) and naltrindole (1.1 x 10(-7) M) prevented the adaptive response development only to the respective agonists. 6. The opioid agonists partially inhibited the spontaneous activity of the tissue. This study has shown that independent activation of mu-, kappa- and delta-opioid receptors can induce dependence in this isolated tissue. Rabbit jejunum is a suitable tissue for studying the acute effects of opioids on the adaptative processes determined by their administration.

Topics: 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; Animals; Atropine; Hexamethonium; Hexamethonium Compounds; In Vitro Techniques; Jejunum; Male; Morphine; Muscle Contraction; Naloxone; Oligopeptides; Pyrrolidines; Rabbits; Receptors, Opioid; Receptors, Opioid, delta; Receptors, Opioid, kappa; Receptors, Opioid, mu; Substance-Related Disorders; Tetrodotoxin