Micelle-associated endomorphin-1 has ability to bind copper in the oxidation state either Cu(II) or Cu(I).

Reduction of Cu(II) to Cu(I) in an oxidizing extracellular environment is a potential risk factor for neurodegenerative diseases, because the re-oxidation of Cu(I) to Cu(II) can be coupled to generation of reactive oxygen species. However, little is known about how the brain is protected from the copper-induced oxidative stress. In the present study, interactions of the endogenous opioid peptide endomorphin-1 (EM1, Tyr-Pro-Trp-Phe-NH2) with ionic copper were investigated. EM1 cannot bind copper with ordinary metal coordination chemistry, since the chelate complex formation of EM1 with the metal ion is inhibited by the proline residue in the second position. In the presence of SDS micelles, however, a significant quenching of fluorescence of the tryptophan side chain of EM1 was observed on addition of copper ion, either Cu(II) or Cu(I). The spectral changes of the UV absorption of the tryptophan, which are diagnostic of cation-π interaction, were also brought about by addition of copper to EM1 only in the presence of micelles. The copper-induced spectral changes of both fluorescence and UV absorption disappeared upon the substitution of Tyr1 with alanine. The obtained results indicated that EM1 binds the copper ion through the π-electrons of aromatic side chains of Tyr1 and Trp3, which are in close contact each other in the micelle-associated form. The copper-catalyzed oxidation/reduction reaction process converting dopamine to neuromelanin, which involves potentially neurotoxic intermediates, is inhibited by EM1. Owing to the ability to bind both Cu(II) and Cu(I), EM1 may have the potential to suppress the copper-mediated oxidative stress in the brain. The present results suggest an antioxidative effect of EM1, distinct from its known analgesic effect.

Preferential Gs protein coupling of the galanin Gal1 receptor in the µ-opioid-Gal1 receptor heterotetramer.

Recent studies have proposed that heteromers of µ-opioid receptors (MORs) and galanin Gal1 receptors (Gal1Rs) localized in the mesencephalon mediate the dopaminergic effects of opioids. The present study reports converging evidence, using a peptide-interfering approach combined with biophysical and biochemical techniques, including total internal reflection fluorescence microscopy, for a predominant homodimeric structure of MOR and Gal1R when expressed individually, and for their preference to form functional heterotetramers when co-expressed. Results show that a heteromerization-dependent change in the Gal1R homodimeric interface leads to a switch in G-protein coupling from inhibitory Gi to stimulatory Gs proteins. The MOR-Gal1R heterotetramer, which is thus bound to Gs via the Gal1R homodimer and Gi via the MOR homodimer, provides the framework for a canonical Gs-Gi antagonist interaction at the adenylyl cyclase level. These novel results shed light on the intense debate about the oligomeric quaternary structure of G protein-coupled receptors, their predilection for heteromer formation, and the resulting functional significance.

Endogenous opioid peptides and brain development: Endomorphin-1 and Nociceptin play a sex-specific role in the control of oligodendrocyte maturation and brain myelination.

The generation of fully functional oligodendrocytes, the myelinating cells of the central nervous system, is preceded by a complex maturational process. We previously showed that the timing of oligodendrocyte differentiation and rat brain myelination were altered by perinatal exposure to buprenorphine and methadone, opioid analogs used for the management of pregnant addicts. Those observations suggested the involvement of the µ-opioid receptor (MOR) and the nociceptin/orphanin FQ receptor (NOR). However, it remained to be determined if these receptors and their endogenous ligands could indeed control the timing of myelination under normal physiological conditions of brain development. We now found that the endogenous MOR ligand endomorphin-1 (EM-1) exerts a striking stimulatory action on cellular and morphological maturation of rat pre-oligodendrocytes, but unexpectedly, these effects appear to be restricted to the cells from the female pups. Critically, this stimulation is abolished by coincubation with the endogenous NOR ligand nociceptin. Furthermore, NOR antagonist treatment of 9-day-old female pups results in accelerated brain myelination. Interestingly, the lack of sex-dependent differences in developmental brain levels of EM-1 and nociceptin, or oligodendroglial expression of MOR and NOR, suggests that the observed sex-specific responses may be highly dependent on important intrinsic differences between the male and female oligodendrocytes. The discovery of a significant effect of EM-1 and nociceptin in the developing female oligodendrocytes and brain myelination, underscores the need for further studies investigating brain sex-related differences and their implications in opioid use and abuse, pain control, and susceptibility and remyelinating capacity in demyelinating disease as multiple sclerosis.

Intracerebroventricular Neuropeptide FF Diminishes the Number of Apneas and Cardiovascular Effects Produced by Opioid Receptors' Activation.

The opioid-induced analgesia is associated with a number of side effects such as addiction, tolerance and respiratory depression. The involvement of neuropeptide FF (NPFF) in modulation of pain perception, opioid-induced tolerance and dependence was well documented in contrast to respiratory depression. Therefore, the aim of the present study was to examine the potency of NPFF to block post-opioid respiratory depression, one of the main adverse effects of opioid therapy. Urethane-chloralose anaesthetized Wistar rats were injected either intravenously (iv) or intracerebroventricularly (icv) with various doses of NPFF prior to iv endomorphin-1 (EM-1) administration. Iv NPFF diminished the number of EM-1-induced apneas without affecting their length and without influence on the EM-1 induced blood pressure decline. Icv pretreatment with NPFF abolished the occurrence of post-EM-1 apneas and reduced also the maximal drop in blood pressure and heart rate. These effects were completely blocked by the NPFF receptor antagonist RF9, which was given as a mixture with NPFF before systemic EM-1 administration. In conclusion, our results showed that centrally administered neuropeptide FF is effective in preventing apnea evoked by stimulation of µ-opioid receptors and the effect was due to activation of central NPFF receptors. Our finding indicates a potential target for reversal of opioid-induced respiratory depression.

Amphiphilic Endomorphin-1 Derivative Functions as Self-assembling Nanomedicine for Effective Brain Delivery.

Endomorphin-1 (Tyr-Pro-Trp-Phe-NH2, EM-1), an endogenous µ-opioid receptor ligand with strong antinociceptive activity, is not in clinical use because of its limited metabolic stability and membrane permeability. In this study, we develop a short-peptide self-delivery system for brain targets with the capability to deliver EM-1 without vehicle. Two amphiphilic EM-1 derivatives, C18-SS-EM1 and C18-CONH-EM1, were synthesized by attaching a stearyl moiety to EM-1 via a disulfide and amide bond, respectively. The amphiphilicity of EM-1 derivatives enabled self-assembling into nanoparticles for brain delivery. The study assessed morphology, circular dichroism, and metabolic stability of the formulations, as well as their pharmacodynamics and in vivo distribution, directly monitored by near-IR fluorescence imaging in mouse brains. In aqueous solution, the C18-SS-EM1 derivative self-assembled into spherical nanostructures with a diameter of 10-20 nm. Near-IR fluorescence analysis visualized the accumulation of the peptides in the brain. Importantly, the analgesic effect of C18-SS-EM1 nanoparticles was significantly stronger as compared to that of unmodified EM-1 or C18-CONH-EM1 nanoparticles. An in vitro release study demonstrated that self-assembled C18-SS-EM1 nanoparticles possessed reduction-responsive behavior. In summary, self-assembling C18-SS-EM1 nanoparticles, which integrate the advantages of lipidization, nanoscale characteristics and, labile disulfide bonds, represent a promising strategy for brain delivery of short peptides.

Effect of Endomorphin-1 on Contractile Activity of Rat Mesenteric Lymphatic Vessels.

We studied the effect of endomorphin-1 on isolated mesenteric lymphatic vessels in rats. It was found that endomorphin-1 caused a dose-dependent increase in the contractile activity of lymphangions, which was associated with stimulation of intracellular calcium depots. The observed effect of endomorphin-1 in isolated lymphatic vessels has a complex mechanism; it depends on the concentration of the applied peptide and is probably determined by its interaction with non-opioid receptors.

Original endomorphin-1 analogues exhibit good analgesic effects.

A new class of endomorphin-1 analogues was synthesized by combining successful chemical modifications including N-terminal guanidino modification, Phe4 was chlorinated, D-Ala-Gly Substituted L-Pro2. Their bioactivities were measured by radioligand binding assay, metabolic stability and the tail-flick test. In radioligand binding assays, analogue GAGPC (Nα-Amidino-Tyr-D-Ala-Gly-Trp-p-Cl-Phe-NH2), shown a µ-opioid receptor affinity about 1.42-fold higher and a 2.51-fold higher δ-opioid receptor affinity than EM-1. In the metabolic stability assays, GAGPC had the longest half-lives which was 284min and 53-fold higher than that of EM-1. In the tail-flick test in mice, GAGPC chloride modification increases the lipid content of the drug, thus increases the permeability of the blood brain barrier, and has a higher analgesic activity. It might be of importance in potential application as drug candidates as analgesic.

Original endomorphin-1 analogues exhibit good analgesic effects with minimal implications for human sperm motility.

To search a novel analgesic characterizes the effects on human sperm motility as minimal as possible. A new class of endomorphin-1 (EM-1) analogues was synthesized by combining successful chemical modifications including N-terminal guanidino modification, Phe4 was chlorinated, replaced of l-Pro2-Trp3 by d-Ala2-Gly3 or d-Pro2-Gly3 at position 2 and 3. Their bioactivities were measured by radioligand binding assay, metabolic stability, antinociception activity and sperm motility effects. In radioligand binding assays, analogue GAGP shown a µ-opioid receptor affinity about 17.7-fold higher and a 57.3-fold higher δ-opioid receptor affinity than EM-1. In the metabolic stability assays, GAGP had the longest half-lives and 16.6-fold higher than EM-1. In the tail-flick test in mice, GAGP showed the best analgesia. In sperm motility assays, the group of GAGP (10-5, 10-7mol/L) decreased of the percentage of a+b grade, and no significant when compared with initial value. In GAGP (10-6mol/L) group, sperm motility was progressively increased, although it was not statistically significant. But at the groups of morphine (10-7mol/L) and GAGD (10-7mol/L), these caused significant reduction between 0 and 90 min. We found that analogues GAGP, activating µ-opioid receptor and partial δ-opioid receptor, exhibit good analgesic effects with minimal implications for human sperm motility. It might be important in potential application as drug candidates of analgesic without implications for human sperm motility.

The role and significance of endomorphin-1 and µ-opioid receptor in rats with endometriosis.

Endomorphin-1 (EM-1) was reported to have very high affinity and selectivity for µ-opioid receptor (MOR). However, it remained unclear whether EM-1 and MOR were involved in the pathologies of endometriosis resulting in reduced fertility. In this study, RT-PCR, radioimmunoassay, immunohistochemistry, and Western blot were used, respectively. The results showed that the immune positive cells of EM-1 in hypothalamus, pituitary, and ovaries were significantly increased in endometriosis model rats, accompanied by the increase of plasma level of EM-1 and the decrease of follicle-stimulating hormone (FSH), luteinizing hormone (LH), estradiol (E2), and progesterone (P). Interestingly, EM-1 was negatively correlated with FSH and LH (p < 0.05). More importantly, Naloxone (MOR antagonist) can significantly reduce the levels of EM-1 in serum, hypothalamus, pituitary, and ovaries, while increased the levels of FSH and LH. In conclusion, our results suggested that EM-1 may be involved in the pathogenesis of the endometriosis-associated infertility by regulating hypothalamus-pituitary-ovarian axis, and Naloxone may be a new alternative drug for the treatment of endometriosis.

Delivery of a lactose derivative of endomorphin 1 to the brain via the olfactory epithelial pathway.

The rapid and direct delivery of a neuroactive endomorphin 1 derivative to the brain via nasal delivery is reported. A synthetic derivative of the native opioid peptide, endomorphin 1 bearing a lactose unit on the N-terminus of the peptide has been previously reported to exhibit antinoceceptive activity similar to morphine after both intravenous and oral administration. This compound has been administered nasally to rats and appeared in the olfactory bulb within 10 min of administration with negligible levels appearing in the circulating blood or in the rest of the brain. These results indicate that the peptide is absorbed into the brain via the olfactory epithelial pathway suggesting nasal delivery may be a viable alternative route of delivery in clinical applications.

Novel Opioids in the Setting of Acute Postoperative Pain: A Narrative Review.

Although traditional opioids such as morphine and oxycodone are commonly used in the management of acute postoperative pain, novel opioids may play a role as alternatives that provide potent pain relief while minimizing adverse effects. In this review, we discuss the mechanisms of action, findings from preclinical studies and clinical trials, and potential advantages of several novel opioids. The more established include oliceridine (biased ligand activity to activate analgesia and downregulate opioid-related adverse events), tapentadol (mu-opioid agonist and norepinephrine reuptake inhibitor), and cebranopadol (mu-opioid agonist with nociceptin opioid peptide activity)-all of which have demonstrated success in the clinical setting when compared to traditional opioids. On the other hand, dinalbuphine sebacate (DNS; semi-synthetic mu partial antagonist and kappa agonist), dual enkephalinase inhibitors (STR-324, PL37, and PL265), and endomorphin-1 analog (CYT-1010) have shown good efficacy in preclinical studies with future plans for clinical trials. Rather than relying solely on mu-opioid receptor agonism to relieve pain and risk opioid-related adverse events (ORAEs), novel opioids make use of alternative mechanisms of action to treat pain while maintaining a safer side-effect profile, such as lower incidence of nausea, vomiting, sedation, and respiratory depression as well as reduced abuse potential.

Dynamic recognition of naloxone, morphine and endomorphin1 in the same pocket of µ-opioid receptors.

Morphine, the most widely used analgesic, relieves severe pain by activating the µ-opioid receptor (MOR), whereas naloxone, with only slight structural changes compared to morphine, exhibits inhibitory effect, and is used to treat opioid abuse. The mechanism by which the MOR distinguishes between the two is unclear. Molecular dynamics (MD) simulations on a 1-µs time scale and metadynamics-enhanced conformational sampling are used here to determine the different interactions of these two ligands with MOR: morphine adjusted its pose by continuously flipping deeper into the pocket, whereas naloxone failed to penetrate deeper because its allyl group conflicts with several residues of MOR. The endogenous peptide ligand endomorphin-1 (EM-1) underwent almost no significant conformational changes during the MD simulations. To validate these processes, we employed GIRK4S143T, a MOR-activated Gßγ-protein effector, in combination with mutagenesis and electrophysiological recordings. We verified the role of some key residues in the dynamic recognition of naloxone and morphine and identified the key residue I322, which leads to differential recognition of morphine and naloxone while assisting EM-1 in activating MOR. Reducing the side chain size of I322 (MORI322A) transformed naloxone from an inhibitor directly into an agonist of MOR, and I322A also significantly attenuated the potency of MOR on EM-1, confirming that binding deep in the pocket is critical for the agonistic effect of MOR. This finding reveals a dynamic mechanism for the response of MOR to different ligands and provides a basis for the discovery of new ligands for MOR at the atomic level.

[PI3K/Akt signaling pathway mediates the protective effect of endomorphin-1 postconditioning against myocardial ischemia-reperfusion injury in rats].

OBJECTIVE: To investigate the role of PI3K/Akt signaling pathway in mediating the protective effect of endomorphin-1 against myocardial ischemia-reperfusion (IR) injury. OBJECTIVE: Fifty SD male rats were randomly divided into sham operation group, myocardial IR group, endomorphin-1 post-treatment group (EM50 group), endomorphin-1+wortmannin (a PI3K/Akt signaling pathway inhibitor) treatment group (EM50+Wort group), and wortmannin treatment group (Wort group). Rat models of myocardial IR injury were established by ligation of the left anterior descending coronary artery for 30 min followed by reperfusion for 120 min. The heart rate and mean arterial pressure were monitored during the experiment. Plasma levels of LDH, CK-MB, cTnI, IL-6, TNF-α, SOD and MDA were measured after reperfusion. The mRNA expression of Bax and Bcl-2 was detected using RT-PCR, and the expression of apoptosis-related protein cleaved caspase-3, phosphorylated Akt protein and total Akt protein in myocardial tissue was detected using Western blotting. OBJECTIVE: Myocardial IR injury significantly decreased heart rate and blood pressure of the rats in comparison with the sham operation (P < 0.05). Compared with those in the IR group, the rats in EM50 group showed significantly increased heart rate and blood pressure (P < 0.05) with decreased plasma LDH, CK-MB, cTnI, IL-6, TNF-α and MDA levels (P < 0.05), increased SOD activity (P < 0.05), increased expression of p-Akt protein and Bcl-2 mRNA (P < 0.05), and decreased expression of Bax mRNA and cleaved caspase-3 protein (P < 0.05). In EM50+Wort group, the heart rate and blood pressure were significantly lowered (P < 0.05), plasma LDH, CK-MB, cTnI, IL-6, TNF-α and MDA levels increased (P < 0.05), SOD activity decreased (P < 0.05), the expression of p-Akt protein and Bcl-2 mRNA was reduced (P < 0.05), and the expression of Bax mRNA and cleaved caspase-3 protein increased (P < 0.05) as compared with those in EM50 group. OBJECTIVE: EM-1 postconditioning can regulate cardiac myocyte apoptosis and reduce myocardial IR injury in rats. The PI3K/Akt signaling pathway may play a role in mediating the myocardial protective effects of EM-1 postconditioning.

Modulation of brain stimulation reward and locomotor activity by ionotropic glutamate receptors of the tail of the ventral tegmental area.

The rostromedial tegmental nucleus also referred to as the tail of the ventral tegmental area (tVTA) contains a cluster of gamma-aminobutyric acid (GABA)ergic neurons that receive dense glutamatergic afferents from the lateral habenula (LHb), and project to dopamine (DA) neurons of the ventral tegmental area (VTA) and substantia nigra pars compacta (SNc). In light of previous evidence implicating glutamate transmission in the regulation of midbrain DA neuronal activity, we first assessed the impact of intra-tVTA microinjection of NBQX (0.8 nmol/side) and PPPA (0.825 nmol/side), respectively AMPA and NMDA receptor antagonists, on reward induced by intracranial self-stimulation (ICSS) and on locomotor activity. Since the tVTA contains a large concentration of mu opioid receptors, additional measures were obtained following microinjection of endomorphin-1 (EM-1, 1 nmol/side) to confirm tVTA placements. Then, using small interfering RNAs (siRNAs), we tested the effect of tVTA downregulation of the GluN1 subunit of the NMDA receptor on reward and locomotor activity. Results show that NBQX, PPPA and EM-1 all enhance reward and locomotor activity, effects that were of different magnitude in rostral and intermediate parts of the tVTA. On the other hand, a reduction in GluN1 subunits used a marked decrease in operant responding for ICSS, but failed to alter ICSS reward and the reward-enhancing effect of PPPA. Our results support a role for the tVTA as a main inhibitory component of DA-dependent behavioral measures, and suggest that tVTA NMDA receptors that modulate reward are most likely expressed on tVTA afferent terminals.

Vagal apnea and hypotension evoked by systemic injection of an antinociceptive analogue of endomorphin-2.

PK20M (Dmt-D-Lys-Phe-Phe-OH) is a novel modified endomorphin-2 (EM-2) peptide producing strong dose- and time-dependent antinociceptive activity. Yet its prototype, endogenous EM-2, has been reported to trigger respiratory and vascular effects such as apnea and hypotension. The purpose of this study was to investigate the potency of the PK20M to evoke respiratory and cardiovascular responses in comparison to endogenous endomorphins. The engagement of the vagal pathway and µ opioid receptors in mediation of these responses was investigated. The effects of intravenous injections of PK20M, EM-1, and EM-2 were studied in anaesthetized, spontaneously breathing rats. The main dose-dependent effect of all endomorphins in the intact rats was immediate apnea, blood pressure and heart rate decrease. PK20M produced apnea in at least half of the intact animals in a much smaller dose than EM-1 and EM-2. The effects of all compounds were abrogated by pre-treatment with MNLX, a peripherally acting µ receptor antagonist. Cervical vagotomy eliminated arrest of breathing in the case of each tested compound. Hypotension was reduced by vagi section only after EM-1 and EM-2 administration. Our results demonstrated that apnea and bradycardia caused by systemic injection of all endomorphins were mediated via activation of µ vagal opioid receptors. The hypotension depended on intact vagi nerves only in the case of EM-1 and EM-2, whereas PK20M decreased blood pressure via other mechanisms outside vagal innervation. Modified opioid agonist is more potent in evoking extended hypotension; at the same time, it produces an arrest of breathing less frequently than its prototype EM-2.

Simultaneous intrathecal injection of muscimol and endomorphin-1 alleviates neuropathic pain in rat model of spinal cord injury.

INTRODUCTION: Due to side effects of medications used for chronic pain, combination therapy seems to be an appropriate solution for alleviation of chronic pain and reducing the side effects. The role of inhibitory GABA system is well proven in reducing neuropathic pain. Also, special attention has been focused on endogenous morphine (endomorphins) in reducing chronic pain originates from damage to the nervous system. The purpose of this study is to investigate the analgesic effect of simultaneous administration of GABA agonist and endomorphin-1 on neuropathic pain in rat model of spinal cord injury (SCI). The role of oxidative stress, NR1 subunits of NMDA receptors, and α2 subunits of GABA receptors in the spinal cord has also been investigated. METHODS: Spinal cord at level of T6-T8 was compressed. Three weeks after spinal cord injury, muscimol and endomorphin-1 were injected (intrathecally once a day for 7 days) individually or in combination. Mechanical and cold allodynia, thermal and mechanical hyperalgesia were evaluated before injection and 15 and 60 min after injection. At the end of behavioral experiments, histological and biochemical evaluations were done on prepared spinal cord samples. RESULTS: Isobologram results showed that combination therapy significantly increased the pain threshold comparing to injection of endomorphin-1 (EM) or muscimol alone. Histological studies indicated the increased expression of α2 subunits of GABA receptors, and NR1 subunits of NMDA receptors in the spinal cord. The combination therapy also increased the glutathione (GSH) and superoxide dismutase (SOD) level and decreased the malondialdehyde (MDA) levels in the spinal cord. CONCLUSION: Simultaneous administration of muscimol and endomorphine-1 could be a new candidate for alleviation of pain resulting from spinal cord injury.

The role of CGRP receptor antagonist (CGRP8-37) and Endomorphin-1 combination therapy on neuropathic pain alleviation and expression of Sigma-1 receptors and antioxidants in rats.

BACKGROUND: Spinal cord injury is one of the most common causes of neuropathic pain which is not responsive to common treatments. Owing to the adverse effects of drugs, it seems that the use of Calcitonin Gene-Related Protein (CGRP) receptor antagonist or Morphine and their combination could be an appropriate strategy for pain alleviation. METHOD: To achieve the objective, fifty six male Wistar rats were divided into seven groups. CGRP8-37 and Endomorphin-1 alone, and in combinated administration, as bolus and continues dose. Both mechanical and cold allodynia, and mechanical hyperalgesia were evaluated before and also15 and 60 min after injection to indicate the efficacy of the therapies in the acute and chronic circumstances on pain induced by spinal cord compression injury. Sigma-1 receptor experssion, oxidant and antioxidant activity after the seven days of the drug adminestration were evaluated. RESULT: The results showed that Endomorphin-1and CGRP8-37 injections were able to reduce neuropathic pain after spinal cord compression injury. Compared to Endomorphin-1, or CGRP8-37 monotherapy, combination therapy did not show more attenuating effects on the pain threshold. Compared to the continous administration of Endomorphin-1 alone, and CGRP8-37 alone, the continous combination therapy did not reduce the pain further. Molecular studies disclosed the increased expression of the Sigma1 receptor, in the spinal cord after administration of Endomorphin-1, and CGRP8-37 alone, as well as combination therapy. Although, an increase in GPx and SOD activity, and decrease in MDA activity was observed in the combination therapy. CONCLUSION: Our results demonstrate that either Endomorphin-1 or CGRP receptor antagonist is able to decrease the neuropathic pain after SCI but combination therapy by a CGRP receptor antagonist and Endomorphin-1 did not make any further reduction in pain sensation.

Green synthesis of bioactive oligopeptides promoted by recyclable nanocrystalline hydroxyapatite.

Aim: The pharmaceutical industry is showing renewed interest in therapeutic peptides. Unfortunately, the chemical synthesis of peptides remains very expensive and problematic in terms of environmental sustainability. Hence, making peptides 'greener' has become a new front line for the expansion of peptide market. Results: We developed a mechanochemical solvent-free peptide bond-forming protocol using standard reagents and nanocrystalline hydroxyapatite as a bio-compatible, reusable inorganic base. The reaction was also conducted under ultra-mild, minimal solvent-grinding conditions, using common laboratory equipment. Conclusion: The efficacy of the described protocol was validated with the convenient preparation of endomorphin-1, H-Tyr-Pro-Trp-Phe-NH2, the endogenous ligand of the µ-opioid receptor, currently regarded as a lead for the discovery of painkillers devoid of harmful side effects.

[Microinjection of endomorphin-1 in the ventrolateral periaqueductal gray induces descending inhibition of cardiac nociception by activating µ-opioid receptor in rats].

OBJECTIVE: To observe descending inhibition of cardiac nociception induced by microinjection of endomorphin-1 (EM1) in the ventrolateral periaqueductal gray (VLPAG) in rats effect and explore the role of µ-opioid receptor in mediating this effect. METHODS: Male SD rats were randomized into electromyography (EMG) group and c-Fos group, both of which were further divided into 5 subgroups, namely 0.9% NaCl group, bradykinin (BK) group, BK+EM1 group, BK+CTOP group, and BK+CTOP+EM1 group. Rat models of cardiac nociception were established by intrapericardial injection of BK. The changes of cardiaosomatic motor reflex induced by BK were observed by assessing EMG responses of the dorsal spinotrapezius muscle; c-Fos expression in the spinal dorsal horn at levels T3-T5 was tested. RESULTS: Compared with 0.9% NaCl, intrapericardial BK injection induced obvious EMG activities and significantly increased c-Fos expression in the spinal dorsal horn at T3-T5 (P < 0.05). Compared with BK injection, microinjection of EM1 in the VLPAG dose-dependently inhibited EMG activities and significantly decreased c-Fos expression (P < 0.05); microinjection of CTOP in the VLPAG produced no significant effect on EMG or c-Fos expression (P > 0.05). Microinjection of CTOP obviously reversed EM1-induced inhibition of EMG activities and c-Fos expression (P < 0.05). CONCLUSIONS: Microinjection of EM1 in the VLPAG produces descending inhibition of cardiac nociception in rats by activating µ-opioid receptor.

Experimental Gene Therapy with Serine-Histogranin and Endomorphin 1 for the Treatment of Chronic Neuropathic Pain.

The insufficient pain relief provided by current pharmacotherapy for chronic neuropathic pain is a serious medical problem. The enhanced glutamate signaling via NMDA receptors appears to be one of the key events in the development of chronic pain. Although effective, clinical use of systemic NMDA antagonists is limited by adverse effects such as hallucinations and motor dysfunction. Opioids are also potent analgesics but their chronic use is accompanied by tolerance and risk of addiction. However, combination of NMDA antagonists and opioids seems to provide a stable pain relieve at subthreshold doses of both substances, eliminating development of side effects. Our previous research showed that combined delivery of NMDA antagonist Serine histrogranin (SHG) and endomorphin1 (EM1) leads to attenuation of acute and chronic pain. The aim of this study was to design and evaluate an analgesic potency of the gene construct encoding SHG and EM1. Constructs with 1SHG copy in combination with EM1, 1SHG/EM1, and 6SHG/EM1 were intraspinally injected to animals with peripheral nerve injury-induced pain (chronic constriction injury, CCI) or spinal cord injury induced pain (clip compression model, SCI) and tactile and cold allodynia were evaluated. AAV2/8 particles were used for gene delivery. The results demonstrated 6SHG/EM1 as the most efficient for alleviation of pain-related behavior. The effect was observed up to 8 weeks in SCI animals, suggesting the lack of tolerance of possible synergistic effect between SHG and EM1. Intrathecal injection of SHG antibody or naloxone attenuated the analgesic effect in treated animals. Biochemical and histochemical evaluation confirmed the presence of both peptides in the spinal tissue. The results of this study showed that the injection of AAV vectors encoding combined SHG/EM constructs can provide long term attenuation of pain without overt adverse side effects. This approach may provide better treatment options for patients suffering from chronic pain.