dynorphins and endomorphin-1

Endomorphin analogs containing unnatural amino acids have demonstrated potent analgesic effects in our previous studies. In the present study, the differences in antinociception and the mechanisms thereof for analogs 1-3 administered intracerebroventricularly and intrathecally were explored. All analogs at different routes of administration produced potent analgesia compared to the parent peptide endomorphin-1. Multiple antagonists and antibodies were used to explore the mechanisms of action of these analogs, and it was inferred that analogs 1-3 stimulated the μ opioid receptor to induce antinociception. Moreover, the antibody data suggested that analog 2 may induce the release of immunoreactive [Leu

Topics: Analgesics; Animals; Chronic Pain; Dose-Response Relationship, Drug; Dynorphins; Enkephalin, Methionine; Enkephalins; Injections, Intraventricular; Mice; Naloxone; Narcotic Antagonists; Oligopeptides; Receptors, Opioid, mu; Spinal Cord

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

Acid-sensing ion channels (ASICs) belong to the DEG/ENaC gene family. While ASIC1a, ASIC1b and ASIC3 are activated by extracellular protons, ASIC4 and the closely related bile acid-sensitive ion channel (BASIC or ASIC5) are orphan receptors. Neuropeptides are important modulators of ASICs. Moreover, related DEG/ENaCs are directly activated by neuropeptides, rendering neuropeptides interesting ligands of ASICs. Here, we performed an unbiased screen of 109 short neuropeptides (<20 amino acids) on five homomeric ASICs: ASIC1a, ASIC1b, ASIC3, ASIC4 and BASIC. This screen revealed no direct agonist of any ASIC but three modulators. First, dynorphin A as a modulator of ASIC1a, which increased currents of partially desensitized channels; second, YFMRFamide as a modulator of ASIC1b and ASIC3, which decreased currents of ASIC1b and slowed desensitization of ASIC1b and ASIC3; and, third, endomorphin-1 as a modulator of ASIC3, which also slowed desensitization. With the exception of YFMRFamide, which, however, is not a mammalian neuropeptide, we identified no new modulator of ASICs. In summary, our screen confirmed some known peptide modulators of ASICs but identified no new peptide ligands of ASICs, suggesting that most short peptides acting as ligands of ASICs are already known.

Topics: Acid Sensing Ion Channels; Animals; Drug Evaluation, Preclinical; Dynorphins; Female; Neuropeptides; Oligopeptides; Sodium Channel Agonists; Xenopus laevis

A novel fritless solid-phase extraction (SPE) microcartridge was designed for combination with sheathless capillary electrophoresis-mass spectrometry (sheathless CE-MS) employing a prototype porous-tip capillary for nanoelectrospray ionization (nanoESI). The inlet of the separation capillary (30μm inner diameter (id), 150μm outer diameter (od)) was inserted in a 4mm long SPE microcartridge (150μm id, 365μm od) packed with a C18 sorbent of 55-105μm particle size. Performance of the SPE-CE-MS system was evaluated using diluted solutions of the three opioid peptides dynorphin A (1-7) (DynA), endomorphin 1 (End1) and met-enkephalin (Met). Sample volumes of 1.5μL were loaded on the SPE microcartridge and the retained peptides were eluted with 22nL of an acidic methanol/water (60:40, v/v) solution. Using a pressure of 50mbar during separation to speed up the analysis, good peptide resolution was obtained with acceptable plate numbers (between 53,000 and 92,000). Intraday relative standard deviations (% RSD) for peptide migration times and peak areas were below 4% and 9%, respectively. The SPE-CE-MS method showed good linearity in the 0.05-5ngmL(-1) range and limits of detection (LODs) were 10pgmL(-1). However, loading a larger volume of sample (8μL), LODs could be decreased down to 2pgmL(-1) (2.2-3.5pM). This represents an improvement of up to 5000-fold with respect to the LODs achieved by sheathless CE-MS without on-line preconcentration demonstrating the potential of on-line SPE for further enhancing sensitivity.

Topics: Dynorphins; Electrophoresis, Capillary; Enkephalin, Methionine; Limit of Detection; Mass Spectrometry; Oligopeptides; Solid Phase Extraction

To observe the analgesic effects of moxibustion in rats with chronic visceral hyperalgesia and its influence on the concentration of dynorphin (Dyn) and endomorphin (EM) in spinal cord.. The rat model of chronic visceral hyperalgesia was established by colorectal distention (CRD). In moxibustion (MX) group, moxibustion was applied once daily for 7 d; in sham moxibustion (SM) group, moxibustion was given to the same acupoints but with the non-smoldered end of the moxa stick. Model control (MC) group and normal control group were also studied. The scoring system of abdominal withdrawal reflex was used to evaluate visceral pain for behavioral assessment. Enzyme linked immunosorbent assay was performed to determine the concentrations of Dyn and EM in spinal cord.. Moxibustion significantly decreased visceral pain to CRD in this rat model, and no significant difference was detected between the SM group and the MC group. In MX group, moxibustion also increased the concentrations of Dyn and EM in spinal cord, and no significant difference was found between the SM group and the MC group.. Moxibustion therapy can significantly enhance the pain threshold of rats with chronic visceral hyperalgesia, and the effect may be closely related to the increased concentration of Dyn and EM in spinal cord.

Topics: Analgesics, Opioid; Animals; Animals, Newborn; Disease Models, Animal; Dynorphins; Humans; Hyperalgesia; Male; Moxibustion; Oligopeptides; Pain Threshold; Rats; Rats, Sprague-Dawley; Reflex, Abdominal; Viscera

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

Capsaicin inhibited the equilibrium specific binding of endogenous opioid-like peptide ligands such as endomorphin-1, nociceptin, and dynorphin((1-17)) in rat brain membrane preparations. We studied the in vitro effect of capsaicin (1-10 microM) on homologous and heterologous competitive binding of opioid ligands, using unlabeled synthetic peptides and the following tritiated compounds: [(3)H]endomorphin-1, [(3)H]endomorphin-2, [(3)H]nociceptin((1-17)) and [(3)H]dynorphin((1-17)). Capsaicin-dependent inhibition was also observed in [(35)S]GTPgammaS stimulation assays in the presence of certain opioid peptides. The inhibition of opioid binding was further investigated using other synthetic and natural mu-opioid ligands such as [D-Ala(2),(NMe)Phe(4),Gly(5)-ol]enkephalin (DAMGO), morphine and naloxone. The decrease in opioid ligand affinity upon capsaicin treatments was most apparent with endomorphin-1, followed by nociceptin and dynorphin. The binding of other investigated opioids were not affected in the presence of capsaicin. In [(3)H]endomorphin-1 binding assays, capsazepine antagonized the inhibitory effect of capsaicin in rat brain membranes suggesting the involvement of TRPV1 receptors. In Chinese hamster ovary (CHO) cells stably expressing mu-opioid receptors, but lacking vanilloid receptors, the inhibition by capsaicin on the binding of [(3)H]endomorphin-1 was not present. It is concluded that the inhibitory effect of capsaicin on the receptor binding affinity of endogenous opioid peptides in brain membrane preparations seems not to be a direct effect, it is rather a negative feedback interaction with opioid receptors.

Topics: Analgesics, Opioid; Animals; Brain; Capsaicin; CHO Cells; Cricetinae; Cricetulus; Dynorphins; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Humans; Ligands; Nociceptin; Nociceptin Receptor; Oligopeptides; Opioid Peptides; Peptides; Protein Binding; Radioligand Assay; Rats; Rats, Wistar; Receptors, Opioid; Receptors, Opioid, kappa; Receptors, Opioid, mu; Sensory System Agents; Tritium

Although opioids are known to influence sleep-wake regulation, the neuroanatomic substrate(s) mediating these effects remain unresolved. We hypothesized that the influence of opiates on sleep may be mediated, at least in part, by the ventrolateral preoptic nucleus (VLPO), a key cell group for producing behavioral sleep. By combining in situ hybridization for kappa and mu receptor mRNA with immunostaining of Fos expressed by VLPO cells during sleep we show that >85% of sleep-active VLPO neurons contain mRNA for either or both opioid receptors. Microinfusions of a kappa receptor agonist into the VLPO region increased NREM sleep by 51% during the subjective night, whereas a mu receptor agonist increased wakefulness by 60% during the subjective day. The sleep- and wake-promoting effects of the kappa and mu agonists were blocked by prior administration of their respective antagonist. Combining retrograde tracing from the VLPO with immunohistochemistry for dynorphin (Dyn, the endogenous kappa receptor agonist) or endomorphin 1 (EM1, the endogenous mu receptor agonist) we show that the central lateral parabrachial subnucleus (PBcl) provides Dyn inputs to the VLPO, whereas hypothalamic histaminergic neurons provide EM1 inputs to the VLPO. In summary, results from the present study suggest that central opioid inputs to the VLPO may play a role in sleep-wake regulation and that the VLPO likely mediates the hypnotic response to high levels of opioid analgesics.

Topics: Analgesics, Opioid; Animals; Dynorphins; Electroencephalography; Hypothalamus; Immunohistochemistry; In Situ Hybridization; Male; Narcotic Antagonists; Neural Pathways; Neurons; Neurotransmitter Agents; Oligopeptides; Preoptic Area; Proto-Oncogene Proteins c-fos; Rats; Rats, Sprague-Dawley; Receptors, Opioid; Receptors, Opioid, kappa; Receptors, Opioid, mu; RNA, Messenger; Sleep; Synaptic Transmission; Wakefulness

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 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

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

Opioid effects on synaptic transmission in the mouse supraoptic nucleus (SON) were investigated using whole-cell, patch-clamp techniques. The mu-opioid receptor agonist, [D-Ala(2), N-Me-Phe(4), Gly(5)-ol]-enkephalin (DAMGO) decreased the amplitude of both evoked excitatory postsynaptic currents (eEPSCs) and inhibitory postsynaptic currents (eIPSCs), and also decreased the frequency of both miniature EPSCs and IPSCs without effect on the amplitude. The selective mu-opioid receptor antagonist, D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH(2), and the nonselective antagonist naloxone, antagonized these inhibitory effects. The application of DAMGO suppressed the amplitude of both the first and second evoked postsynaptic currents with a paired-pulse stimulus protocol, but increased the paired-pulse ratios (second ePSC/first ePSC). DAMGO induced neither inward nor outward currents, and had no significant changes in either glutamate- or GABA-induced currents. When compared with the relatively selective kappa- and delta-opioid receptor agonists dynorphin and [D-Pen(2), D-Pen(5)]-enkephalin, DAMGO showed the most potent inhibitory effects on evoked and miniature postsynaptic currents. Taken together, these results imply that DAMGO strongly suppresses the release of glutamate and GABA via mu-opioid receptors in the mouse SON, and support the involvement of presynaptic regulation by opioids in the control of magnocellular neurosecretory neurones.

Topics: Analgesics, Opioid; Animals; Dynorphins; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, D-Penicillamine (2,5)-; Excitatory Postsynaptic Potentials; gamma-Aminobutyric Acid; Glutamic Acid; Hypothalamus; Male; Mice; Mice, Inbred ICR; Neural Inhibition; Neurons; Oligopeptides; Organ Culture Techniques; Patch-Clamp Techniques; Receptors, Opioid; Supraoptic Nucleus

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

Opioid peptides have been detected in the auditory and vestibular efferent neurons where they colocalize with the major neurotransmitter, acetylcholine. We investigated the function of opioids to modulate neurotransmission mediated by hair cell's alpha9/alpha10-containing nicotinic acetylcholine receptors (alpha9/alpha10nAChRs). The endogenous opioid peptides, endomorphin-1 (mu agonist) and dynorphin B (kappa agonist), but not a delta agonist [D-Pen2,D-Pen-5]enkephalin, inhibited the acetylcholine-evoked currents in frog saccular hair cells and rat inner hair cells. This inhibition was noncompetitive, voltage-independent, and was accompanied by an acceleration of the rate of current decay. Selective mu- and kappa-opioid receptor antagonists did not block the inhibition, although partial reduction by naloxone was observed. All opioid antagonists tested also reduced the acetylcholine response. Endomorphin-1 and dynorphin B inhibited the acetylcholine-evoked currents in alpha9/alpha10-expressing Xenopus oocytes. Because oocytes lack opioid receptors, it provides strong evidence for the direct interaction of opioid peptides with alpha9/alpha10nAChR.. alpha9/alpha10nAChR is a target for modulation by endomorphin-1 and dynorphin B, efferent cotransmitters in the inner ear.

Topics: Acetylcholine; Animals; Anura; Cochlea; Dynorphins; Ear, Inner; Electric Conductivity; Endorphins; Enkephalin, D-Penicillamine (2,5)-; Hair Cells, Auditory; In Vitro Techniques; Narcotic Antagonists; Neurotransmitter Agents; Oligopeptides; Oocytes; Protein Isoforms; Rats; Rats, Sprague-Dawley; Receptors, Nicotinic; Saccule and Utricle; Synapses; Xenopus laevis

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