Endomorphin-1 Research Articles

Mexneurin is a novel precursor of peptides in the central nervous system of rodents

Endomorphins (EMs) have been proposed as the endogenous ligand agonists of the μ-opioid receptor; however, no propeptide precursor protein for EMs has been identified. Here, to identify the presumed precursor of EMs, we designed an immunoscreening assay using specific affinity-purified rabbit antisera raised against synthetic EMs in a whole-mouse brain cDNA library. Following this approach, we identify a DNA sequence encoding a protein precursor, which we name proMexneurin, that contains three different peptide sequences: Mexneurin-1 (an EM-like peptide), Mexneurin-2, and Mexneurin-3, a peptide which appears to be unrelated to EMs. RT-PCR analysis and in situ hybridization reveal a widespread distribution of proMexneurin mRNA throughout the mouse brain. Both Mexneurin-1 and Mexneurin-3 peptides display biological activities in the mouse CNS.

Decreased spinal endomorphin-2 contributes to mechanical allodynia in streptozotocin-induced diabetic rats

Diabetic neuropathic pain (DNP) plays a major role in decreased life quality of diabetes patients, however, the neural mechanisms underlying DNP remain unclear. Endomorphins are the endogenous ligands for mu-opioid receptor. There is increasing evidence implicating the involvement of spinal endomorphin-2 (EM2) in neuropathic pain. In this study, using a streptozotocin induced diabetic rat model that displayed obvious mechanical allodynia, it was found that the expression of spinal EM2 was significantly decreased in DNP rats. While intrathecal administration of exogenous EM2 attenuated mechanical allodynia in DNP rats, the mu-opioid receptor antagonist β-funaltrexamine facilitated these events. It was found that the reduction in spinal EM2 was mediated by increased activity of dipeptidylpeptidase IV, possibly as a consequence of diabetes-induced oxidative stress. Taken together, our results provide the first evidence that the reduction in the level of an endogenous opioid in primary afferents was significantly associated with DNP. This indicates that the chronic pain associated with DNP might be due to the loss of an inhibitory effect on pain signal transmission.

Targeting ASIC3 for Relieving Mice Fibromyalgia Pain: Roles of Electroacupuncture, Opioid, and Adenosine

Many scientists are seeking better therapies for treating fibromyalgia (FM) pain. We used a mouse model of FM to determine if ASIC3 and its relevant signaling pathway participated in FM pain. We demonstrated that FM-induced mechanical hyperalgesia was attenuated by electroacupuncture (EA). The decrease in fatigue-induced lower motor function in FM mice was also reversed by EA. These EA-based effects were abolished by the opioid receptor antagonist naloxone and the adenosine A1 receptor antagonist rolofylline. Administration of opioid receptor agonist endomorphin (EM) or adenosine A1 receptor agonist N6-cyclopentyladenosine (CPA) has similar results to EA. Similar results were also observed in ASIC3−/− or ASIC3 antagonist (APETx2) injected mice. Using western blotting, we determined that pPKA, pPI3K, and pERK were increased during a dual acidic injection priming period. Nociceptive receptors, such as ASIC3, Nav1.7, and Nav1.8, were upregulated in the dorsal root ganglion (DRG) and spinal cord (SC) of FM mice. Furthermore, pPKA, pPI3K, and pERK were increased in the central thalamus. These aforementioned mechanisms were completely abolished in ASIC3 knockout mice. Electrophysiological results also indicated that acid potentiated Nav currents through ASIC3 and ERK pathway. Our results highlight the crucial role of ASIC3-mediated mechanisms in the treatment of FM-induced mechanical hyperalgesia.

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.

Distribution of endomorphin-like-immunoreactive neurones in the brain of the cichlid fish Oreochromis mossambicus.

Endomorphins (EMs) are tetrapeptides involved in pain and neuroendocrine responses with a high affinity for μ-opioid receptors in mammals. In the present study, we investigated the distribution of EM-like-immunoreactive (EM-L-IR) neurones in the brain of the cichlid fish Oreochromis mossambicus. Application of antisera against EM-1 and 2 (EM-1-2) revealed the presence of EM-L-IR somata and fibres throughout the different subdivisions of the olfactory bulb, such as the olfactory nerve layer and the granule cell layer. Although the extensions of EM-L-IR fibres were seen along the medial olfactory tract, intensely labelled EM-L-IR somata were found in different subdivisions of the telencephalon. In the diencephalon, intensely stained EM-L-IR neurones were noted in the preoptic area, the nucleus preopticus pars magnocellularis, the suprachiasmatic nucleus, the nucleus lateralis tuberis pars lateralis and the nucleus lateralis tuberis pars medialis regions, whereas projections of EM-L-IR fibres were also seen along the hypothalamic-hypophyseal tract, suggesting a possible hypophysiotrophic role for these neurones. Intense to moderately stained EM-L-IR neurones were noted in different subdivisions of thalamic nucleus, such as the dorsal posterior thalamic nucleus, commissura posterior, ventromedial thalamic nucleus, nucleus posterior tuberis, ventrolateral thalamic nucleus and medial preglomerular nucleus. Numerous intensely stained perikarya and axonal fibres were also noted throughout the inferior lobe, along the periventricular margin of the reccessus lateralis and in the nucleus recesus lateralis regions. In addition, numerous moderately labelled EM-like neuronal populations were found in the secondary gustatory nucleus and rostral spinal cord. The widespread distribution of EM-L-IR neurones throughout the brain and spinal cord indicates the diverse roles for these cells in neuroendocrine and neuromodulatory responses for the first time in fish. The present study provides further insights into the possible existence of EM-like peptides in early vertebrate lines and suggests that these peptides might have been well-conserved during the course of evolution.

Electroacupuncture Attenuates CFA-induced Inflammatory Pain by suppressing Nav1.8 through S100B, TRPV1, Opioid, and Adenosine Pathways in Mice

Pain is associated with several conditions, such as inflammation, that result from altered peripheral nerve properties. Electroacupuncture (EA) is a common Chinese clinical medical technology used for pain management. Using an inflammatory pain mouse model, we investigated the effects of EA on the regulation of neurons, microglia, and related molecules. Complete Freund’s adjuvant (CFA) injections produced a significant mechanical and thermal hyperalgesia that was reversed by EA or a transient receptor potential V1 (TRPV1) gene deletion. The expression of the astrocytic marker glial fibrillary acidic protein (GFAP), the microglial marker Iba-1, S100B, receptor for advanced glycation end-products (RAGE), TRPV1, and other related molecules was dramatically increased in the dorsal root ganglion (DRG) and spinal cord dorsal horn (SCDH) of CFA-treated mice. This effect was reversed by EA and TRPV1 gene deletion. In addition, endomorphin (EM) and N6-cyclopentyladenosine (CPA) administration reliably reduced mechanical and thermal hyperalgesia, thereby suggesting the involvement of opioid and adenosine receptors. Furthermore, blocking of opioid and adenosine A1 receptors reversed the analgesic effects of EA. Our study illustrates the substantial therapeutic effects of EA against inflammatory pain and provides a novel and detailed mechanism underlying EA-mediated analgesia via neuronal and non-neuronal pathways.

Sequence-Specific Regulation of Endocytic Lifetimes Modulates Arrestin-Mediated Signaling at the µ Opioid Receptor

Functional selectivity at the µ opioid receptor (µR), a prototypical G-protein-coupled receptor that is a physiologically relevant target for endogenous opioid neurotransmitters and analgesics, has been a major focus for drug discovery in the recent past. Functional selectivity is a cumulative effect of the magnitudes of individual signaling pathways, e.g., the Gαi-mediated and the arrestin-mediated pathways for µR. The present work tested the hypothesis that lifetimes of agonist-induced receptor-arrestin clusters at the cell surface control the magnitude of arrestin signaling, and therefore functional selectivity, at µR. We show that endomorphin-2 (EM2), an arrestin-biased ligand for µR, lengthens surface lifetimes of receptor-arrestin clusters significantly compared with morphine. The lengthening of lifetimes required two specific leucines on the C-terminal tail of µR. Mutation of these leucines to alanines decreased the magnitude of arrestin-mediated signaling by EM2 without affecting G-protein signaling, suggesting that lengthened endocytic lifetimes were required for arrestin-biased signaling by EM2. Lengthening surface lifetimes by pharmacologically slowing endocytosis was sufficient to increase arrestin-mediated signaling by both EM2 and the clinically relevant agonist morphine. Our findings show that distinct ligands can leverage specific sequence elements on µR to regulate receptor endocytic lifetimes and the magnitude of arrestin-mediated signaling, and implicate these sequences as important determinants of functional selectivity in the opioid system.

Endomorphin-2 Inhibition of Substance P Signaling within Lamina I of the Spinal Cord Is Impaired in Diabetic Neuropathic Pain Rats.

Opiate analgesia in the spinal cord is impaired in diabetic neuropathic pain (DNP), but until now the reason is unknown. We hypothesized that it resulted from a decreased inhibition of substance P (SP) signaling within the dorsal horn of the spinal cord. To investigate this possibility, we evaluated the effects of endomorphin-2 (EM2), an endogenous ligand of the μ-opioid receptor (MOR), on SP release within lamina I of the spinal dorsal horn (SDH) in rats with DNP. We established the DNP rat model and compared the analgesic efficacy of EM2 between inflammation pain and DNP rat models. Behavioral results suggested that the analgesic efficacy of EM2 was compromised in the condition of painful diabetic neuropathy. Then, we measured presynaptic SP release induced by different stimulating modalities via neurokinin-1 receptor (NK1R) internalization. Although there was no significant change in basal and evoked SP release between control and DNP rats, EM2 failed to inhibit SP release by noxious mechanical and thermal stimuli in DNP but not in control and inflammation pain model. We also observed that EM2 decreased the number of FOS-positive neurons within lamina I of the SDH but did not change the amount of FOS/NK1R double-labeled neurons. Finally, we identified a remarkable decrease in MORs within the primary afferent fibers and dorsal root ganglion (DRG) neurons by Western blot (WB) and immunohistochemistry (IHC). Taken together, these data suggest that reduced presynaptic MOR expression might account for the loss of the inhibitory effect of EM2 on SP signaling, which might be one of the neurobiological foundations for decreased opioid efficacy in the treatment of DNP.

Decreased Endomorphin-2 and μ-Opioid Receptor in the Spinal Cord Are Associated with Painful Diabetic Neuropathy.

Painful diabetic neuropathy (PDN) is one of the most common complications in the early stage of diabetes mellitus (DM). Endomorphin-2 (EM2) selectively activates the μ-opioid receptor (MOR) and subsequently induces antinociceptive effects in the spinal dorsal horn. However, the effects of EM2-MOR in PDN have not yet been clarified in the spinal dorsal horn. Therefore, we aimed to explore the role of EM2-MOR in the pathogenesis of PDN. The main findings were the following: (1) streptozotocin (STZ)-induced diabetic rats exhibited hyperglycemia, body weight loss and mechanical allodynia; (2) in the spinal dorsal horn, the expression levels of EM2 and MOR decreased in diabetic rats; (3) EM2 protein concentrations decreased in the brain, lumbar spinal cord and cerebrospinal fluid (CSF) in diabetic rats but were unchanged in the plasma; (4) the frequency but not the amplitude of spontaneous excitatory postsynaptic currents (sEPSCs) was significantly higher in diabetic rats than in control rats; and (5) intrathecal injection of EM2 for 14 days in the early stage of PDN partially alleviated mechanical allodynia and reduced MOR expression in diabetic rats. Our results demonstrate that the EM2-MOR signal may be involved in the early stage of PDN.

Effects of endomorphin-1 postconditioning on myocardial ischemia/reperfusion injury and myocardial cell apoptosis in a rat model.

Endomorphins (EMs) have important roles in the body with regards to analgesia, feeding behavior, gastrointestinal movement and inflammatory reaction. Recent studies have reported that EMs may also participate in chronic hypoxia in the protection of rat myocardial ischemia/reperfusion; however, the mediator and underlying mechanisms remain to be elucidated. The aim of the present study was to investigate the effects of EM‑1 postconditioning on myocardial ischemia/reperfusion injury (MIRI) and myocardial cell apoptosis in a rat model, and to assess its likely mechanisms. A total of 48male Sprague Dawley rats were randomly divided into four groups: Sham group, ischemia/reperfusion group (IR group), ischemic postconditioning group (IPO group) and EM‑1 postconditioning group (EM50 group). A MIRI model was established via occlusion of the left anterior descending branch of the coronary artery for 30min, followed by reperfusion for 120min invivo. Hemodynamic indexes were recorded and analyzed. Following reperfusion, plasma lactate dehydrogenase (LDH), creatine kinase‑MB (CK‑MB), malondialdehyde (MDA), superoxide dismutase (SOD), interleukin‑6 (IL‑6) and tumor necrosis factor‑α (TNF‑α) contents or activities were measured, infarct size was determined, and the expression levels of B-cell lymphoma2 (Bcl-2) and Bcl-2-associated X protein (Bax) mRNA and cleaved caspase‑3 protein were assessed. In the IR group, mean arterial pressure (MAP) and heart rate (HR) were decreased compared with in the sham group. In addition, LDH and CK‑MB levels were increased; IL‑6, TNF‑α and MDA content was increased; SOD activity was decreased; the Bcl‑2/Bax ratio was decreased; and cleaved caspase‑3 protein expression levels were increased in the IR group. Compared with in the IR group, in the IPO and EM50 groups, MAP and heart rate (HR) were recovered to various extents post‑reperfusion; LDH and CK‑MB levels were decreased; IL‑6, TNF‑α and MDA content was decreased; SOD activity was increased; infarct size was reduced; the Bcl‑2/Bax ratio was increased; and cleaved caspase‑3 protein expression levels were decreased. In conclusion, EM‑1 postconditioning was revealed to reduce I/R injury and inhibit myocardial cell apoptosis, which may be associated with reductions in oxidative stress and inflammatory reactions.

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.

Pharmacological characterization of EN-9, a novel chimeric peptide of endomorphin-2 and neuropeptide FF that produces potent antinociceptive activity and limited tolerance

Mounting evidences indicate the functional interactions between neuropeptide FF (NPFF) and opioids, including the endogenous opioids. In the present work, EN-9, a chimeric peptide containing the functional domains of the endogenous opioid endomorphin-2 (EM-2) and NPFF, was synthesized and pharmacologically characterized. In vitro cAMP assay demonstrated that EN-9 was a multifunctional agonist of κ-opioid, NPFF1 and NPFF2 receptors. In the mouse tail-flick test, intracerebroventricularly (i.c.v.) administration of EN-9 produced significant antinociception with an ED50 value of 13.44 nmol, which lasted longer than that of EM-2. In addition, EN-9 induced potent antinociception after both intravenous (i.v.) and subcutaneous (s.c.) injection. Furthermore, the experiments using the antagonists of opioid and NPFF receptors indicated that the central antinociception of EN-9 was mainly mediated by κ-opioid receptor, independently on NPFF receptors. Notably, the central antinociception of EN-9 was not reduced over a period of 6 days repeated i.c.v. injection. Repeated i.c.v. administration of EN-9 with the NPFF1 and NPFF2 receptors antagonist RF9 resulted in a progressive loss of analgesic potency, consistent with the development of tolerance. Moreover, central administration of EN-9 induced the place conditioning aversion only at a high dose of 60 nmol, but not at low doses. At supraspinal level, only high dose of EN-9 (60 nmol, i.c.v.) inhibited gastrointestinal transit via NPFF receptors. Similarly, systemic administration of EN-9 also inhibited gastrointestinal transit at high doses (10 and 30 mg/kg, i.v.). Taken together, the multifunctional agonist of κ-opioid and NPFF receptors EN-9 produced a potent, non-tolerance forming antinociception with limited side effects.

Inhibitory Effect of Endomorphin-2 Binding to the μ-Opioid Receptor in the Rat Pre-Bötzinger Complex on the Breathing Activity.

Opiates are commonly used analgesics that often cause clinical respiratory depression. However, their underlying mechanisms remain unclear. Endomorphin-2 (EM2) is a novel, endogenous tetrapeptide opioid with very high affinity and selectivity for the μ-opioid receptor (MOR). The pre-Bötzinger complex (pre-BötC) is considered the center of respiratory rhythm generation, and the synaptic connections in this region are essential for respiratory rhythm. The present study identified EM2-like immunoreactive (LI) axonal terminals in the pre-BötC of adult rats. Some EM2-LI axonal terminals made principally symmetric synapses with neurokinin 1 receptor (NK1R)-LI or MOR-LI neuronal dendritic processes in the pre-BötC. Unilateral microinjection of EM2 into the pre-BötC decreased breathing frequency and amplitude. A prior microinjection of the selective MOR antagonist β-funaltrexamine (β-FNA) into the pre-BötC prevented the effects of EM2. The present results suggest that EM2-LI axonal terminals modulate NK1R-expressing neurons in the pre-BötC and that EM2 plays a role in respiratory depression through MORs in the pre-BötC.

Synthesis of mixed MOR/KOR efficacy cyclic opioid peptide analogs with antinociceptive activity after systemic administration

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.

P3Neural circuits for pain modulation in the central nervous system

Pain is a common and severe disease that affects millions of people worldwide and is difficult to treat. The main reason is that the mechanisms for pain information transmission and modulation have still not revealed. In the central nervous system (CNS), there are many long and short neural circuits constituted by axonal processes and are necessary for pain sensation. But the structures and functions of these pathways, especially those constituted by local axons and their collaterals are not known, which is essential for us to uncover the neural mechanisms of pain [1]. In our recent studies, the neural circuits involved in pain sensation in the CNS have been investigated with different methods, especially under the electron microscopic level [2]. The main results are as follows: (1) In the superficial laminae (lamina I and lamina II) of the spinal dorsal horn (SDH), substance P-immunoreactive (SP-ir) and calcitonin gene-related peptide (CGRP)-ir primary afferent terminals have been observed to make synapses with GABA- and glycine-ir neurons which could be activated by periphery painful stimulation, suggesting that the noxious information might be regulated by inhibitory interneurons in the SDH. (2) Endomorphin (EM) is a new member of the endogenous opioid family and specific ligand for μ-opioid receptor (MOR) [3]. Within the superficial laminae of the SDH, either EM-ir fibers from the primary afferents (Figure 1) or serotonin (5-HT)-ir fibers from the descending fibers of the brainstem raphe nuclei have been encountered to terminate onto the same neurons which send projection fibers to the parabrachial nucleus (PBN) or ventrolateral complex of the thalamus, indicating that the SDH might be a very special site for pain modulation. (3) Neurons in layer V of the anterior cingulate cortex (ACC) innervated by direct ascending fibers from the spinal dorsal horn, and then in turn, projected directly to the SDH. The excitation of these ACC-SDH projection neurons by nociceptive inputs could facilitate the activity of the nociceptive neurons in lamina I of the SDH, suggesting that the descending projection from ACC to SDH might directly potentiate the noxious information transmission. This effect might affect the patient and make them to feel much more painful [4]. (4) In the periaqueductal gray, EM released from the terminals originated from the EM-containing neurons in the hypothalamus binds to MOR expressing GABAeregic inhibitory interneurons and then results in disinhibition of 5-HTergic projection neurons, and finally to exert analgesic effects at the spinal cord level [5]. These results have provided morphological and functional evidence for the mechanisms underlying pain information transmission and modulation in the CNS.

Endomorphin-1 attenuates Aβ42 induced impairment of novel object and object location recognition tasks in mice

A growing body of evidence suggests that the agglomeration of amyloid-β (Aβ) may be a trigger for Alzheimer׳s disease (AD). Central infusion of Aβ42 can lead to memory impairment in mice. Inhibiting the aggregation of Aβ has been considered a therapeutic strategy for AD. Endomorphin-1 (EM-1), an endogenous agonist of μ-opioid receptors, has been shown to inhibit the aggregation of Aβ in vitro. In the present study, we investigated whether EM-1 could alleviate the memory-impairing effects of Aβ42 in mice using novel object recognition (NOR) and object location recognition (OLR) tasks. We showed that co-administration of EM-1 was able to ameliorate Aβ42-induced amnesia in the lateral ventricle and the hippocampus, and these effects could not be inhibited by naloxone, an antagonist of μ-opioid receptors. Infusion of EM-1 or naloxone separately into the lateral ventricle had no influence on memory in the tasks. These results suggested that EM-1 might be effective as a drug for AD preventative treatment by inhibiting Aβ aggregation directly as a molecular modifier.

Design, synthesis, and evaluation of new endomorphin analogs with enhanced central antinociception after peripheral administration.

We synthesized two novel endomorphin-1 (EM-1) analogs by substituting the C-terminus residue with (thienyl)-α-methylene-β-amino acids (Map). Several in vitro and in vivo assays were used to determine the activity of the analogs. The two EM-1 analogs showed subnanomolar binding affinity and functional activity at the μ-opioid receptor in HEK293 cells. Tail-flick and formalin tests further revealed that the EM-1 analogs were very effective after intravenous administration. Our results indicate that compared to endomorphin-1, the (thienyl)Map modified peptides showed improved blood-brain barrier permeability.

Contribution of Endogenous Spinal Endomorphin 2 to Intrathecal Opioid Antinociception in Rats Is Agonist Dependent and Sexually Dimorphic

Interactions between exogenous and endogenous opioids are not commonly investigated as a basis for sexually dimorphic opioid analgesia. We investigated the influence of spinal endomorphin 2 (EM2), an endogenous mu-opioid receptor (MOR) ligand, on the spinal antinociception produced by intrathecally administered opioids. Activation of spinal MORs facilitated spinal EM2 release. This effect was sexually dimorphic, occurring in males but not in females. Although activational effects of testosterone were required for opioid facilitation of spinal EM2 release in males, the absence of this facilitation in females did not result from either insufficient levels of testosterone or mitigating effects of estrogens. Strikingly, in males, the contribution of spinal EM2 to the analgesia produced by intrathecally applied MOR agonists depended on their analgesic efficacy relative to that of EM2. Spinal EM2 released by the higher efficacy MOR agonist sufentanil diminished sufentanil's analgesic effect, whereas EM2 released by the lower efficacy morphine had the opposite effect on spinal morphine antinociception. Understanding antithetical contributions of endogenous EM2 to intrathecal opioid antinociception not only enlightens the selection of opioid medications for pain management but also helps to explain variable sex dependence of the antinociception produced by different opioids, facilitating the acceptance of sexually dimorphic antinociception as a basic tenet. PerspectiveThe male-specific MOR-coupled enhancement of spinal EM2 release implies a parallel ability to harness endogenous EM2 antinociception. The inferred diminished ability of females to utilize the spinal EM2 antinociceptive system could contribute to their greater frequency and severity of chronic pain syndromes.

Novel endomorphin-1 analogs with C-terminal oligoarginine-conjugation display systemic antinociceptive activity with less gastrointestinal side effects.

In recent study, in order to improve the bioavailability of endomorphin-1 (EM-1), we designed and synthesized a series of novel EM-1 analogs by replacement of L-Pro(2) by β-Pro, D-Ala or Sar, together with C-terminal oligoarginine-conjugation. Our results indicated that the introduction of D-Ala and β-Pro in position 2, along with oligoarginine-conjugation, didn't significantly decrease the μ-affinity and in vitro bioactivity, and the enhancement of arginine residues did not markedly influence the μ-affinity of these analogs. All analogs displayed a significant enhancement of stability, which may be due to increased resistance to proline-specific enzymatic degradation. Moreover, following intracerebroventricular (i.c.v.) administration, analogs 1, 2, 4 and 5 produced significant antinociception and increased duration of action, with the ED50 values being about 1.8- to 4.2-fold less potent than that of EM-1. In addition, our results indicated that no significant antinociceptive activity of EM-1 was seen following subcutaneous (s.c.) injection, whereas analogs 1, 2, 4 and 5 with equimolar dose induced significant and prolonged antinociception by an opioid and central mechanism. Herein, we further examined the gastrointestinal transit and colonic propulsive latencies of EM-1 and its four analogs administered centrally and peripherally. I.c.v. administration of EM-1 and analogs 1, 2, 4 and 5 significantly delayed gastrointestinal transit and colonic bead propulsion in mice, but the inhibitory effects induced by these analogs were largely attenuated. It is noteworthy that no significant gastrointestinal side effects induced by these four analogs were observed after s.c. administration. Our results demonstrated that combined modifications of EM-1 with unnatural amino acid substitutions and oligoarginine-conjugation gave an efficient strategy to improve the analgesic profile of EM-1 analogs but with less gastrointestinal side effects when administered peripherally.

Inhibitory effects of endomorphin-2 on excitatory synaptic transmission and the neuronal excitability of sacral parasympathetic preganglionic neurons in young rats.

The function of the urinary bladder is partly controlled by parasympathetic preganglionic neurons (PPNs) of the sacral parasympathetic nucleus (SPN). Our recent work demonstrated that endomorphin-2 (EM-2)-immunoreactive (IR) terminals form synapses with μ-opioid receptor (MOR)-expressing PPNs in the rat SPN. Here, we examined the effects of EM-2 on excitatory synaptic transmission and the neuronal excitability of the PPNs in young rats (24–30 days old) using a whole-cell patch-clamp approach. PPNs were identified by retrograde labeling with the fluorescent tracer tetramethylrhodamine-dextran (TMR). EM-2 (3 μM) markedly decreased both the amplitude and the frequency of the spontaneous and miniature excitatory postsynaptic currents (sEPSCs and mEPSCs) of PPNs. EM-2 not only decreased the resting membrane potentials (RMPs) in 61.1% of the examined PPNs with half-maximal response at the concentration of 0.282 μM, but also increased the rheobase current and reduced the repetitive action potential firing of PPNs. Analysis of the current–voltage relationship revealed that the EM-2-induced current was reversed at −95 ± 2.5 mV and was suppressed by perfusion of the potassium channel blockers 4-aminopyridine (4-AP) or BaCl2 or by the addition of guanosine 5′-[β-thio]diphosphate trilithium salt (GDP-β-S) to the pipette solution, suggesting the involvement of the G-protein-coupled inwardly rectifying potassium (GIRK) channel. The above EM-2-invoked inhibitory effects were abolished by the MOR selective antagonist D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2 (CTOP), indicating that the effects of EM-2 on PPNs were mediated by MOR via pre- and/or post-synaptic mechanisms. EM-2 activated pre- and post-synaptic MORs, inhibiting excitatory neurotransmitter release from the presynaptic terminals and decreasing the excitability of PPNs due to hyperpolarization of their membrane potentials, respectively. These inhibitory effects of EM-2 on PPNs at the spinal cord level may explain the mechanism of action of morphine treatment and morphine-induced bladder dysfunction in the clinic.