dynorphins and endomorphin-2

Opioid agonist activation at the mu opioid receptor (MOR) can lead to a wide variety of physiological responses. Many opioid agonists share the ability to selectively and preferentially activate specific signaling pathways, a term called biased agonism. Biased opioid ligands can theoretically induce specific physiological responses and might enable the generation of drugs with improved side effect profiles.. Dynorphins, enkephalins, and endomorphins are endogenous opioid agonist peptides that may possess distinct bias profiles; biased agonism of endogenous peptides could explain the selective roles of these ligands in vivo. Our purpose in the present study was to investigate biased signaling and potential underlying molecular mechanisms of bias using. We found that endomorphin-1/2 preferentially activated cAMP signaling, while dynorphin-B preferentially activated. We found that endomorphin-1/2 and dynorphin-B displayed contrasting bias profiles at the MOR, and ruled out potential AC6 and RGS4 mechanisms in this bias. This identified signaling bias could be involved in specifying endogenous peptide roles in vivo, where these peptides have low selectivity between opioid receptor family members.

Topics: Adenylyl Cyclases; Animals; Cell Culture Techniques; Cell Line; CHO Cells; Cricetulus; Cyclic AMP; Dynorphins; Endorphins; Gene Knockdown Techniques; Guanosine 5'-O-(3-Thiotriphosphate); Humans; Oligopeptides; Receptors, Opioid, mu; RGS Proteins; Signal Transduction

Cyclic pentapeptide Tyr-c[D-Lys-Phe-Phe-Asp]NH2, based on the structure of endomorphin-2 (EM-2), which shows high affinity to the μ-opioid receptor (MOR) and a very strong antinociceptive activity in mice was used as a parent compound for the structure-activity relationship studies. In this report we synthesized analogs of a general sequence Dmt-c[D-Lys-Xaa-Yaa-Asp]NH2, with D-1- or D-2-naphthyl-3-alanine (D-1-Nal or D-2-Nal) in positions 3 or 4. In our earlier papers we have indicated that replacing a phenylalanine residue by the more extended aromatic system of naphthylalanines may result in increased bioactivities of linear analogs. The data obtained here showed that only cyclopeptides modified in position 4 retained the sub-nanomolar MOR and nanomolar κ-opioid receptor (KOR) affinity, similar but not better than that of a parent cyclopeptide. In the in vivo mouse hot-plate test, the most potent analog, Dmt-c[D-Lys-Phe-D-1-Nal-Asp]NH2, exhibited higher than EM-2 but slightly lower than the cyclic parent peptide antinociceptive activity after peripheral (ip) and also central administration (icv). Conformational analyses in a biomimetic environment and molecular docking studies disclosed the structural determinants responsible for the different pharmacological profiles of position 3- versus position 4-modified analogs.

Topics: Amino Acid Sequence; Analgesics, Opioid; Animals; CHO Cells; Cricetulus; Guinea Pigs; Male; Mice; Molecular Docking Simulation; Oligopeptides; Pain; Peptides, Cyclic; Rats, Wistar; Receptors, Opioid, kappa; Receptors, Opioid, mu; Structure-Activity Relationship

Tachykinin and opioid peptides play a central role in pain transmission, modulation and inhibition. The treatment of pain is very important in medicine and many studies using NK1 receptor antagonists failed to show significant analgesic effects in humans. Recent investigations suggest that both pronociceptive tachykinins and the analgesic opioid systems are important for normal pain sensation. The analysis of opioid peptides in Tac1(-/-) spinal cord tissues offers a great opportunity to verify the influence of the tachykinin system on specific opioid peptides. The objectives of this study were to develop an HPLC-MS/MRM assay to quantify targeted peptides in spinal cord tissues. Secondly, we wanted to verify if the Tac1(-/-) mouse endogenous opioid system is hampered and therefore affects significantly the pain modulatory pathways. Targeted neuropeptides were analyzed by high performance liquid chromatography linear ion trap mass spectrometry. Our results reveal that EM-2, Leu-Enk and Dyn A were down-regulated in Tac1(-/-) spinal cord tissues. Interestingly, Dyn A was almost 3 fold down-regulated (p<0.0001). No significant concentration differences were observed in mouse Tac1(-/-) spinal cords for Met-Enk and CGRP. The analysis of Tac1(-/-) mouse spinal cords revealed noteworthy decreases of EM-2, Leu-Enk and Dyn A concentrations which strongly suggest a significant impact on the endogenous pain-relieving mechanisms. These observations may have insightful impact on future analgesic drug developments and therapeutic strategies.

Topics: Animals; Chromatography, Liquid; Dynorphins; Enkephalin, Leucine; Male; Mice; Mice, Inbred C57BL; Neuropeptides; Oligopeptides; Spectrometry, Mass, Electrospray Ionization; Spinal Cord; Tachykinins

Multiple opioid receptor (OR) types and endogenous opioid peptides exist in the spinal dorsal horn and there may be interactions among these receptor types that involve opioid peptides. In a previous study we observed that antinociceptive effects of the selective κ-opioid receptor (κOR) agonist, U50,488H, was attenuated in μ-opioid receptor (μOR) knockout mice as compared to wild-type mice when administered spinally. This suggests that an interaction between κORs and μORs exits in the spinal cord. The present study was aimed at investigating whether endogenous opioid peptides were involved in such interaction.. We examined whether the presence of antibodies to endogenous opioid peptides, endomorphin-2, met-enkephalin and dynorphin A affected the antinociceptive effects of spinal U50,488H in rats. The tail-flick test was used to assess pain thresholds.. The increase in tail-flick latency after spinal U50,488H was attenuated when the rats were pretreated intrathecally with antiserum against endomorphin-2. Pretreatments with antisera against met-enkephalin and dynorphin A had no effect on U50,488H antinociception. The antisera alone did not affect pain threshold.. The results suggest that endomorphin-2, an endogenous opioid peptide highly selective to the μOR, plays a role in antinociception induced by κOR activation in the spinal cord.

Topics: 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; Analgesics, Non-Narcotic; Animals; Dynorphins; Enkephalin, Methionine; Male; Oligopeptides; Pain; Rats; Rats, Sprague-Dawley; Receptors, Opioid, kappa; Receptors, Opioid, mu; Spinal Cord

Sexually dimorphic nociception and opioid antinociception is very pervasive but poorly understood. We had demonstrated that spinal morphine antinociception in females, but not males, requires the concomitant activation of spinal μ- and κ-opioid receptors (MOR and KOR, respectively). This finding suggests an interrelationship between MOR and KOR in females that is not manifest in males. Here, we show that expression of a MOR/KOR heterodimer is vastly more prevalent in the spinal cord of proestrous vs. diestrous females and vs. males. Cross-linking experiments in combination with in vivo pharmacological analyses indicate that heterodimeric MOR/KOR utilizes spinal dynorphin 1-17 as a substrate and is likely to be the molecular transducer for the female-specific KOR component of spinal morphine antinociception. The activation of KOR within the heterodimeric MOR/KOR provides a mechanism for recruiting spinal KOR-mediated antinociception without activating the concomitant pronociceptive functions that monomeric KOR also subserves. Spinal cord MOR/KOR heterodimers represent a unique pharmacological target for female-specific pain control.

Topics: Analgesia; Analgesics, Opioid; Animals; Dynorphins; Estrous Cycle; Female; Injections, Spinal; Male; Morphine; Nociceptors; Oligopeptides; Protein Binding; Protein Multimerization; Rats; Rats, Sprague-Dawley; Receptors, Opioid, kappa; Receptors, Opioid, mu; Sex Characteristics; Spinal Cord

A method using capillary liquid chromatography-triple-stage mass spectrometry (LC-MS(3)) to determine endogenous opioid peptides in microdialysis samples collected in vivo was developed, validated, and applied to measurements in the rat striatum. Peptides in dialysate rapidly degraded when stored at room temperature or -80 degrees C. Adding acetic acid to a final concentration of 5% stabilized the peptides for 5 days allowing storage of fractions and off-line measurements which proved more convenient and reliable than previously used on-line methods. Study of the effect of dialysis flow rate from 0.2 to 2 microL/min and column inner diameter (i.d.) from 25 to 75 microm on the relative signal obtained for peptides revealed that lowest flow rates and smallest column i.d. gave the highest relative signal. The method was tested for 10 different neuropeptides and limits of detection (LODs) were from 0.5 to 60 pM (4 microL samples) for most. beta-Endorphin had an LOD of 5 nM when detected directly, but it could be quantitatively determined by detecting a characteristic peptide produced by tryptic digestion with an LOD of 3 pM. This approach may prove useful for other large neuropeptides as well. The method was used to determine met-enkephalin, leu-enkephalin, dynorphin A(1-8), and beta-endorphin in vivo. Endomorphin 1 and 2 were below the detection limit of the method in vivo. Quantitative determination of leu-enkephalin using external calibration was verified by standard addition experiments. The improvements over previous approaches using capillary LC-MS(n) make in vivo neuropeptide monitoring more practical and feasible for a variety of neuropeptides.

Topics: Animals; beta-Endorphin; Chromatography, High Pressure Liquid; Corpus Striatum; Dynorphins; Enkephalin, Leucine; Enkephalin, Methionine; Limit of Detection; Male; Microdialysis; Neuropeptides; Oligopeptides; Rats; Rats, Sprague-Dawley; Spectrometry, Mass, Electrospray Ionization

It has been demonstrated that the antinociception induced by i.t. or i.c.v. administration of endomorphins is mediated through mu-opioid receptors. Moreover, though endomorphins do not have appreciable affinity for kappa-opioid receptors, pretreatment with the kappa-opioid receptor antagonist nor-binaltorphimine markedly blocks the antinociception induced by i.c.v.- or i.t.-injected endomorphin-2, but not endomorphin-1. These evidences propose the hypothesis that endomorphin-2 may initially stimulate the mu-opioid receptors, which subsequently induces the release of dynorphins acting on kappa-opioid receptors to produce antinociception. The present study was performed to determine whether the release of dynorphins by i.c.v.-administered endomorphin-2 is mediated through mu-opioid receptors for producing antinociception. Intracerebroventricular pretreatment with an antiserum against dynorphin A, but not dynorphin B or alpha-neo-endorphin, and s.c. pretreatment with kappa-opioid receptor antagonist nor-binaltorphimine dose-dependently attenuated the antinociception induced by i.c.v.-administered endomorphin-2, but not endomorphin-1 and DAMGO. The attenuation of endomorphin-2-induced antinociception by pretreatment with antiserum against dynorphin A or nor-binaltorphimine was dose-dependently eliminated by additional s.c. pretreatment with a selective mu-opioid receptor antagonist beta-funaltrexamine or a selective mu1-opioid receptor antagonist naloxonazine at ultra low doses, which are inactive against micro-opioid receptor agonists in antinociception, suggesting that endomorphin-2 stimulates distinct subclass of micro1-opioid receptor that induces the release of dynorphin A acting on kappa-opioid receptors in the brain. It concludes that the antinociception induced by supraspinally administered endomorphin-2 is in part mediated through the release of endogenous kappa-opioid peptide dynorphin A, which is caused by the stimulation of distinct subclass of micro1-opioid receptor.

Topics: Analgesics; Animals; Dynorphins; Endorphins; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Immune Sera; Injections, Intraventricular; Male; Mice; Naloxone; Naltrexone; Oligopeptides; Protein Precursors; Receptors, Opioid, kappa; Receptors, Opioid, mu

To pursue further the possible de novo biosynthetic pathway of endomorphins in rat brain we raised antibodies to endomorphin-2 conjugate in rabbits. Antiserum R1 recognized endomorphin-2 with good selectivity as compared to endomorphin-1 with a median detection value of 65.5+/-7.5 pg/tube (n=7), whereas R4 antiserum recognized both endomorphins with similar sensitivity. Neither antisera recognized YP-related di- or tripeptides or YGGF-related opioid sequences (enkephalins, beta-endorphin, dynorphin). Using the same rat brain extraction-RP-HPLC-gradient separation paradigm as previously, antisera detected 144.6+/-40.0 (n=3) pg/g wet brain weight endomorphin-2-like immunoreactivity in the fraction corresponding to standard endomorphin-2 retention time and also in the fraction matching endomorphin-2-OH standard retention time (179.1+/-30.1 pg/g). Since R1 failed to recognize authentic endomorphin-2-OH, the second immunoreactive species must be different from both endomorphin-2 and endomorphin-2-OH. Possible biosynthetic intermediates to endomorphins, synthetic YPFFG and YPWFG had retention times close to the parent endomorphin standards in RP-HPLC gradient separation profile. The former was a mu-opioid receptor agonist of medium potency in the in vitro assays (rat brain RBA>P gamma S binding and mouse vas deferens), whereas the latter was a weak mu-opioid receptor agonist with a significant delta-opioid receptorial action as well and a definite indication of partial agonism.

Topics: Amino Acid Sequence; Animals; beta-Endorphin; Brain; Chromatography, High Pressure Liquid; Dynorphins; Enkephalins; Immune Sera; Male; Mice; Narcotic Antagonists; Oligopeptides; Peptides; Rabbits; Radioimmunoassay; Rats; Rats, Wistar

The effects of endomorphin-2 or endomorphin-1 microinjected into the centromedial amygdala on the thermally-induced tail-flick response were studied in male CD rats. Microinjection of endomorphin-2 (8.7-35.0 nmol) given into the centromedial amygdala time- and dose-dependently decreased the tail-flick latencies. On the other hand, endomorphin-1 (8-32.6 nmol) given into the same site did not cause any change of the tail-flick latency. However, endomorphin-1 (32.6 nmol) or endomorphin-2 (35.0 nmol) given into the basolateral site of amygdala did not affect the tail-flick latency. Pretreatment with the antiserum against dynorphin A(1-17) (200 microg) significantly reversed the decrease of the tail-flick latency induced by endomorphin-2. The decrease of the tail-flick latency induced by endomorphin-2 was also blocked by the endomorphin-2 selective micro-opioid receptor antagonist 3-methoxynaltrexone (6.4 pmol) and by the N-methyl-D-aspartate (NMDA) receptor antagonist MK-801 (30 nmol), but not by the kappa-opioid receptor antagonist nor-binaltorphimine (6.6 nmol). It is concluded that endomorphin-2, but not endomorphin-1, given into the centromedial amygdala stimulates a 3-methoxynaltrexone-sensitive mu-opioid receptor subtype to induce the release of dynorphin A(1-17), which then acts on the NMDA receptor, but not kappa-opioid receptor for producing hyperalgesia. This conclusion is further supported by the additional findings that dynorphin A(1-17) (2.3 nmol) given into the centromedial amygdala also caused the decrease of the tail-flick latency, which was similarly blocked by the NMDA receptor antagonist MK-801 (30 nmol), but not kappa-opioid receptor antagonist nor-binaltorphimine (6.6 nmol).

Topics: Amygdala; Analgesics, Opioid; Animals; Dizocilpine Maleate; Dose-Response Relationship, Drug; Dynorphins; Excitatory Amino Acid Antagonists; Hyperalgesia; Immune Sera; Male; Microinjections; Naltrexone; Narcotic Antagonists; Oligopeptides; Pain Measurement; Rabbits; Rats; Receptors, Opioid, mu; Time Factors

The antinociception induced by i.t. or i.c.v. administration of endomorphins is mediated via mu-opioid receptors. However, although endomorphins do not have an appreciable affinity for kappa-opioid receptors, pretreatment with the kappa-opioid receptor antagonist norbinaltorphimine markedly reduces the antinociceptive response to i.c.v. or i.t. administered endomorphin-2 but not endomorphin-1. These results suggest that endomorphin-2 initially stimulates mu-opioid receptors, which subsequently induce the release of dynorphins that act on kappa-opioid receptors to produce antinociception. The present study was performed in mice to determine whether the release of dynorphins by i.t. administered endomorphin-2 is mediated through mu-opioid receptors to produce antinociception. Intrathecal pretreatment with an antiserum against dynorphin A-(1-17), but not against dynorphin B-(1-13) or alpha-neoendorphin, dose-dependently prevented the paw-withdrawal inhibition by endomorphin-2. The pretreatments with these antisera did not affect the endomorphin-1- or [D-Ala(2),MePhe(4),Gly(ol)(5)]enkephalin-induced paw-withdrawal inhibition. The attenuation of endomorphin-2-induced antinociception by i.t. pretreatment with an antiserum against dynorphin A-(1-17) or s.c. pretreatment with norbinaltorphimine was blocked dose-dependently by s.c. pretreatment with the mu-opioid receptor antagonist beta-funaltrexamine or the mu(1)-opioid receptor antagonist naloxonazine at ultra-low doses that are ineffective against mu-opioid receptor agonists. These results suggest that the spinal antinociception induced by endomorphin-2 is mediated through the stimulation of a distinct subtype of mu(1)-opioid receptor that induces the release of the endogenous kappa-opioid peptide dynorphin A-(1-17) in the spinal cord.

Topics: Analgesics, Opioid; Animals; Dose-Response Relationship, Drug; Dynorphins; Hot Temperature; Immune Sera; Injections, Spinal; Male; Mice; Mice, Inbred Strains; Oligopeptides; Pain; Pain Measurement; Physical Stimulation; Receptors, Opioid, mu; Time Factors

The effects of pretreatment with endomorphin-1 (EM-1) and endomorphin-2 (EM-2) given into the ventral periaqueductal gray (vPAG) to induce antianalgesia against the tail-flick (TF) inhibition produced by morphine given into the vPAG were studied in rats. Pretreatment with EM-1 (3.5-28 nmol) given into vPAG for 45 min dose-dependently attenuated the TF inhibition produced by morphine (9 nmol) given into vPAG. Similarly, pretreatment with EM-2 (1.7-7.0 nmol) for 45 min also attenuated the TF inhibition induced by morphine; however, a high dose of EM-2 (14 nmol) did not attenuate the morphine-produced TF inhibition. The attenuation of morphine-produced TF inhibition induced by EM-2 or EM-1 pretreatment was blocked by pretreatment with mu-opioid antagonist (-)-naloxone (55 pmol) but not nonopioid (+)-naloxone (55 pmol). However, pretreatment with a morphine-6beta-glucuronide-sensitive mu-opioid receptor antagonist 3-methoxynaltrexone (6.4 pmol) selectively blocked EM-2- but not EM-1-induced antianalgesia. Pretreatment with dynorphin A(1-17) antiserum reversed only EM-2- but not EM-1-induced antianalgesia. Pretreatment with antiserum against beta-endorphin, [Met(5)]enkephalin, [Leu(5)]enkephalin, substance P or cholecystokinin, or with delta-opioid receptor antagonist naltrindole (2.2 nmol) or kappa-opioid receptor antagonist norbinaltorphimine (6.6 nmol) did not affect EM-2-induced antianalgesia. It is concluded that EM-2 selectively releases dynorphin A(1-17) by stimulation of a novel subtype of mu-opioid receptor, tentatively designated as mu(3) in the vPAG to induce antianalgesia, whereas the antianalgesia induced by EM-1 is mediated by the stimulation of another subtype of mu(1)- or mu(2)-opioid receptor.

Topics: Analgesia; Animals; beta-Endorphin; Cholecystokinin; Dose-Response Relationship, Drug; Dynorphins; Enkephalin, Leucine; Enkephalin, Methionine; Male; Morphine; Naloxone; Oligopeptides; Periaqueductal Gray; Rats; Receptors, Opioid, mu; Substance P

Increased endogenous opioid activity has been implicated in cholestatic pruritus. In the present study, we have further defined the involvement of opioids in cholestasis. Rats underwent either bile duct ligation or a sham operation. Five days after surgery, brains were removed and agonist-stimulated [35S]GTPgammaS binding was measured in ten brain regions. Serum endomorphin-2, leu-enkephalin and dynorphin A levels were measured using ELISA on day five. Microdialysis to the dorsal hypothalamic area was conducted in the same animal before and after cholestasis. Dialysate endomorphin-1, leu-enkephalin and dynorphin A levels also were measured. Delta- and kappa-stimulated binding was significantly decreased in cholestasic animals compared to controls in the dorsal hypothalamic area. The serum dynorphin A level was lower in the cholestasic group than in controls (2.56+/-0.09 and 3.29+/-0.22 ng/ml, respectively, P<0.01). We propose that pruritus in cholestasis may result from an impaired balance between mu- and kappa-opioid systems.

Topics: Animals; Binding, Competitive; Brain; Cholestasis; Dialysis Solutions; Disease Models, Animal; Dynorphins; Enkephalin, Leucine; Guanosine 5'-O-(3-Thiotriphosphate); Hypothalamus; Male; Microdialysis; Oligopeptides; Rats; Rats, Sprague-Dawley; Receptors, Opioid, kappa; Sulfur Radioisotopes

An unbiased conditioned place preference (CPP) paradigm was used to evaluate the reward effects of endogenous mu-opioid receptor ligands endomorphin-1 (EM-1) and endomorphin-2 (EM-2) from the mesolimbic posterior nucleus accumbens (Acb) shell and the ventral tegmental area (VTA) in CD rats. EM-1 (1.6-8.1 nmol) microinjected into posterior Acb shell produced CPP, whereas EM-2 (8.7-17.5 nmol) given into the same Acb shell produced conditioned place aversion (CPA). EM-1 (1.6-16.3 nmol) microinjected into the VTA produced CPP, whereas EM-2 (8.7 and 17.5 nmol) given into the same VTA site did not produce any effect, but at a high dose (35 nmol) produced CPP. EM-1 (3.3 nmol) or EM-2 (17.5 nmol) microinjected into the nigrostriatal substantia nigra was not significantly different from vehicle-injected groups. D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH(2) (CTOP) at 94.13 pmol or 3-methoxynaltrexone at 0.64 pmol microinjected into the posterior Acb shell blocked EM-1-induced CPP and EM-2-induced CPA. At a higher dose, CTOP (941.3 pmol) and 3-methoxynaltrexone (6.4 pmol) produced CPA and CPP, respectively. Coadministration with antiserum against dynorphin A(1-17) (Dyn) (10 microg) microinjected into the posterior Acb shell blocked EM-2-induced CPA. However, it did not affect EM-1-induced CPP. It is concluded that EM-1 and EM-2 produce site-dependent CPP and CPA, respectively, by stimulation of different subtypes of mu-opioid-receptors; stimulation of one subtype of mu-opioid-receptor at the posterior Acb shell and VTA by EM-1 induces CPP, whereas stimulation of another subtype of mu-opioid receptor at the posterior Acb shell, but not the VTA, by EM-2 induces the release of Dyn to produce CPA.

Topics: Animals; Conditioning, Psychological; Dynorphins; Male; Microinjections; Naltrexone; Nucleus Accumbens; Oligopeptides; Rats; Receptors, Opioid, kappa; Receptors, Opioid, mu; Serum; Somatostatin; Space Perception; Substantia Nigra; Ventral Tegmental Area

Intrathecal (i.t.) pretreatment with a low dose (0.3 nmol) of morphine causes an attenuation of i.t. morphine-produced analgesia; the phenomenon has been defined as morphine-induced antianalgesia. The opioid-produced analgesia was measured with the tail-flick (TF) test in male CD-1 mice. Intrathecal pretreatment with low dose (0.3 nmol) of morphine time dependently attenuated i.t. morphine-produced (3.0 nmol) TF inhibition and reached a maximal effect at 45 min. Intrathecal pretreatment with morphine (0.009-0.3 nmol) for 45 min also dose dependently attenuated morphine-produced TF inhibition. The i.t. morphine-induced antianalgesia was dose dependently blocked by the nonselective mu-opioid receptor antagonist (-)-naloxone and by its nonopioid enantiomer (+)-naloxone, but not by endomorphin-2-sensitive mu-opioid receptor antagonist 3-methoxynaltrexone. Blockade of delta-opioid receptors, kappa-opioid receptors, and N-methyl-D-aspartate (NMDA) receptors by i.t. pretreatment with naltrindole, nor-binaltorphimine, and (-)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate (MK-801), respectively, did not affect the i.t. morphine-induced antianalgesia. Intrathecal pretreatment with antiserum against dynorphin A(1-17), [Leu]-enkephalin, [Met]-enkephalin, beta-endorphin, cholecystokinin, or substance P also did not affect the i.t. morphine-induced antianalgesia. The i.t. morphine pretreatment also attenuated the TF inhibition produced by opioid muagonist [D-Ala2, N-Me-Phe4,Gly-ol5]-enkephalin, delta-agonist deltorphin II, and kappa-agonist U50,488H. It is concluded that low doses (0.009-0.3 nmol) of morphine given i.t. activate an antianalgesic system to attenuate opioid mu-, delta-, and kappa-agonist-produced analgesia. The morphine-induced antianalgesia is not mediated by the stimulation of opioid mu-, delta-, or kappa-receptors or NMDA receptors. Neuropeptides such as dynorphin A(1-17), [Leu]-enkephalin, [Met]-enkephalin, beta-endorphin, cholecystokinin, and substance P are not involved in this low-dose morphine-induced antianalgesia.

Topics: Analgesia; Animals; beta-Endorphin; Dizocilpine Maleate; Drug Interactions; Drug Tolerance; Dynorphins; Enkephalins; Male; Mice; Morphine; Naloxone; Naltrexone; Oligopeptides; Pain; Pain Measurement; Receptors, Opioid, delta; Receptors, Opioid, kappa; Receptors, Opioid, mu; Spinal Cord; Substance P

We previously demonstrated pretreatment with antiserum against dynorphin A1-17 attenuates endomorphin-2-induced analgesia and antianalgesia, suggesting that these endomorphin-2 effects are mediated by the release of dynorphin A1-17. Lumbar-cisternal spinal perfusion was used to measure the release of immunoreactive dynorphin A1-17 into spinal perfusates from urethane-anesthetized rats following endomorphin-2 or endomorphin-1 treatment within the perfusion solution. Treatment with endomorphin-2 (5-50 nmol) for 3 min caused a dose-dependent increase of immunoreactive dynorphin A1-17 in spinal perfusates, with a maximal increase detected between 24 and 48 min after endomorphin-2 treatment, while levels returned to baseline within 60 min. Endomorphin-2-induced release of immunoreactive dynorphin A1-17 was attenuated by pretreatment with mu-opioid receptor antagonist naloxone or 3-methoxynaltrexone. Endomorphin-1 induced a slight increase in immunoreactive dynorphin1-17 as well, but only at the highest dose used (50 nmol). Our results suggest that endomorphin-2 stimulated a specific subtype of mu-opioid receptor to induce the release of immunoreactive dynorphin A1-17 in spinal cords of rats.

Topics: Anesthesia; Animals; Dose-Response Relationship, Drug; Dynorphins; Enzyme-Linked Immunosorbent Assay; Injections, Spinal; Male; Naloxone; Narcotic Antagonists; Oligopeptides; Rats; Receptors, Opioid, mu; Spinal Cord

We have previously demonstrated that both endomorphin-1 (EM-1) and endomorphin-2 (EM-2) at high doses (1.75-35 nmol) given intrathecally (i.t.) or intracerebroventricularly produce antinociception by stimulation of mu-opioid receptors. Now, we report that EM-2 at small doses (0.05-1.75 nmol), which injected alone did not produce antinociception, produces anti-analgesia against opioid agonist-induced antinociception. The tail-flick (TF) response was used to test the antinociception in male CD-1 mice. Intrathecal pretreatment with EM-2 (0.02-1.75 nmol) 45 min before i.t. morphine (3.0 nmol) injection dose dependently attenuated morphine-induced TF inhibition. On the other hand, a similar dose of EM-1 (1.64 nmol) failed to produce any antianalgesic effect. The EM-2 (1.75 nmol)-produced anti-analgesia against morphine-induced TF inhibition was blocked by i.t. pretreatment with the mu-opioid antagonist naloxone or 3-methoxynaltrexone, but not delta-opioid receptor antagonist naltrindole, kappa-opioid receptor antagonist nor-binaltorphimine, or N-methyl-d-aspartate (NMDA) receptor antagonist MK-801. The EM-2-induced antianalgesic effect against morphine-induced TF inhibition was blocked by i.t. pretreatment with antiserum against dynorphin A(1-17), but not beta-endorphin, [Met]-enkephalin, [Leu]-enkephalin, or cholecystokinin antiserum (200 microg each). The i.t. EM-2 pretreatment also attenuated the TF inhibition induced by other mu-opioid agonists, [d-Ala2,N-Me-Phe4,Gly-ol5]-enkephalin, EM-1 and EM-2, delta-opioid agonist deltorphin II, and kappa-opioid agonist (trans)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl]benzeneacetamide methane-sulfonate hydrate (U50,488H). It is concluded that EM-2 at subanalgesic doses presumably stimulates a subtype of mu-opioid receptor and subsequently induces the release of dynorphin A(1-17) to produce antianalgesic effects against mu-, delta-, or kappa-agonists-induced antinociception. The EM-2-induced antianalgesia is not mediated by the release of [Met]-enkephalin, [Leu]-enkephalin, beta-endorphin, or cholecystokinin, nor does it involve kappa- or delta-opioid or NMDA receptors in the spinal cord.

Topics: Analgesics, Opioid; Animals; Antibodies, Blocking; Dizocilpine Maleate; Dose-Response Relationship, Drug; Dynorphins; Excitatory Amino Acid Antagonists; Injections, Spinal; Male; Mice; Morphine; Naloxone; Naltrexone; Narcotic Antagonists; Oligopeptides; Pain Measurement; Reaction Time; Receptors, Opioid; Spinal Cord

Two highly selective mu-opioid receptor agonists, endomorphin-1 and endomorphin-2, have been identified and postulated to be endogenous ligands for mu-opioid receptors. Intrathecal (i.t.) administration of endomorphin-1 and endomorphin-2 at doses from 0.039 to 5 nmol dose-dependently produced antinociception with the paw-withdrawal test. The paw-withdrawal inhibition rapidly reached its peak at 1 min, rapidly declined and returned to the pre-injection levels in 20 min. The inhibition of the paw-withdrawal responses to endomorphin-1 and endomorphin-2 at a dose of 5 nmol observed at 1 and 5 min after injection was blocked by pretreatment with a non-selective opioid receptor antagonist naloxone (1 mg/kg, s.c.). The antinociceptive effect of endomorphin-2 was more sensitive to the mu (1)-opioid receptor antagonist, naloxonazine than that of endomorphin-1. The endomorphin-2-induced paw-withdrawal inhibition at both 1 and 5 min after injection was blocked by pretreatment with kappa-opioid receptor antagonist nor-binaltorphimine (10 mg/kg, s.c.) or the delta(2)-opioid receptor antagonist naltriben (0.6 mg/kg, s.c.) but not the delta(1)-opioid receptor antagonist 7-benzylidine naltrexone (BNTX) (0.6 mg/kg s.c.). In contrast, the paw-withdrawal inhibition induced by endomorphin-1 observed at both 1 and 5 min after injection was not blocked by naloxonazine (35 mg/kg, s.c.), nor-binaltorphimine (10 mg/kg, s.c.), naltriben (0.6 mg/kg, s.c.) or BNTX (0.6 mg/kg s.c.). The endomorphin-2-induced paw-withdrawal inhibition was blocked by the pretreatment with an antiserum against dynorphin A-(1-17) or [Met(5)]enkephalin, but not by antiserum against dynorphin B-(1-13). Pretreatment with these antisera did not affect the endomorphin-1-induced paw-withdrawal inhibition. Our results indicate that endomorphin-2 given i.t. produces its antinociceptive effects via the stimulation of mu (1)-opioid receptors (naloxonazine-sensitive site) in the spinal cord. The antinociception induced by endomophin-2 contains additional components, which are mediated by the release of dynorphin A-(1-17) and [Met(5)]enkephalin which subsequently act on kappa-opioid receptors and delta(2)-opioid receptors to produce antinociception.

Topics: Analgesics; Animals; Benzylidene Compounds; Dose-Response Relationship, Drug; Dynorphins; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, Leucine; Enkephalin, Methionine; Immune Sera; Injections, Spinal; Injections, Subcutaneous; Male; Mice; Naloxone; Naltrexone; Narcotic Antagonists; Oligopeptides; Pain; Pain Measurement; Pain Threshold; Peptide Fragments; Time Factors

Long-term memory formation for passive-avoidance learning in the day-old chick is known to have two distinct time windows of protein synthesis (F.M. Freeman, S.P.R. Rose, A.B. Scholey, 1995. Two time windows of anisomycin-induced amnesia for passive-avoidance training in the day-old chick. Neurobiol. Learn. Mem. 63, 291-295). The lobus parolfactorius (LPO) is thought to be an important site for the second wave of protein synthesis which occurs 4-5 h after training. Birds received bilateral intracranial injections of agonists and antagonists for the mu-, delta-, kappa-opioid receptors and the opioid receptor-like (ORL(1)) receptor directly into the LPO at 5 h post-training and were tested for recall 24 h later. Also, 100 microM beta-funaltrexamine (beta-FAN), a mu-opioid receptor antagonist, significantly impaired memory formation (P<0.01). The delta-opioid receptor was also involved in memory formation at this time-point since antagonism of this receptor by 1 mM ICI-174,864 caused amnesia (P<0.01) which was reversed by the agonist, DPLPE. The kappa-opioid receptor appeared not to be involved during the second phase of neuronal activity since neither stimulation by dynorphin nor inhibition by nor-BIN caused amnesia for the task. The ORL(1) receptor agonist orphanin FQ also had no effect suggesting that this receptor was not involved at this 5-h time-point. Cytosolic and mitochondrial protein synthesis has been shown to be important in passive-avoidance learning in the day-old chick. Both chloramphenicol (CAP) and anisomycin (ANI), inhibitors of mitochondrial and cytosolic protein synthesis, respectively, caused disruption when injected 5 h post-training into the LPO (P<0.05). Endomorphin-2 (Endo-2), a mu-opioid receptor agonist, reversed both the ANI- and CAP-sensitivity. However, DPLPE, a delta-opioid receptor agonist, only reversed the effect due to CAP. Possible mechanisms for these effects are discussed.

Topics: Age Factors; Amnesia; Analgesics, Opioid; Animals; Anisomycin; Avoidance Learning; Brain Chemistry; Chickens; Chloramphenicol; Conditioning, Psychological; Dynorphins; Enkephalin, D-Penicillamine (2,5)-; Enkephalin, Leucine; Female; Male; Memory; Naltrexone; Narcotic Antagonists; Neurons; Nociceptin; Oligopeptides; Opioid Peptides; Protein Synthesis Inhibitors; Receptors, Opioid; Receptors, Opioid, delta; Receptors, Opioid, kappa; Receptors, Opioid, mu; Vasodilator Agents