Endogenous Opioid Agonist Research Articles

Examining The Effects Of Gallein On Opioid Induced GIRK Currents

Opioids are powerful analgesics, but carry a risk of overdose that has become a pressing issue to public health in the United States. Overdose deaths are caused by the opioid-induced shutdown of neuronal circuits that operate unconscious breathing cycles, leading to respiratory depression. Respiratory depression, alongside antinociception, is mediated by the activation of μ-opioid receptors, which are coupled to inhibitory heterotrimeric G proteins. Recent studies have shown that gallein, a Gβγ subunit inhibitor, amplifies the antinociceptive effects of opioids through Gq-phospholipase C (PLC) mediated mechanisms, while either attenuating or having no effect on respiratory depression. The molecular mechanisms of gallein effects on respiratory depression are unclear. Based on the binding interface, gallein is predicted to inhibit Gβγ binding to phospholipase C, but not G protein inwardly rectifying potassium channels (GIRK). The goal of this study is to examine the effects of gallein on opioid-induced GIRK currents, with or without Gq subunit activation, in mouse locus coeruleus (LC) neurons. The LC is an area of particular interest due its involvement in respiratory circuits, and activation of Gq-coupled pathways decreases the amplitude of opioid-mediated GIRK currents in LC neurons. We hypothesized that gallein would have no effect on opioid mediated GIRK currents without Gq activation, but would prevent Gq-mediated inhibition of GIRK. We tested this hypothesis using whole-cell voltage-clamp recordings of LC neurons contained in brain slices from adult male and female mice. The endogenous opioid agonist Met-enkephalin induced outward GIRK currents that were not significantly altered by gallein. The Gq-coupled receptor agonist oxotremorine-M reduced the amplitude of Met-enkephalin mediated GIRK currents, as expected. Preliminary data suggests no effect of gallein on GIRK currents in the presence of Gq activation, contrary to our hypothesis. These results will increase our understanding of the role of PLC in Gq-mediated inhibition of GIRK currents at the cellular level and provide insight regarding the potential respiratory related impacts associated with using gallein derived compounds in combination with opioids to increase opioid antinociception and reduce the doses of opioids needed to treat pain. This research is supported by NIH Grant R01DA047978-05 to ESL. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Nav1.7 is essential for nociceptor action potentials in the mouse in a manner independent of endogenous opioids

Loss-of-function mutations in Nav1.7, a voltage-gated sodium channel, cause congenital insensitivity to pain (CIP) in humans, demonstrating that Nav1.7 is essential for the perception of pain. However, the mechanism by which loss of Nav1.7 results in insensitivity to pain is not entirely clear. It has been suggested that loss of Nav1.7 induces overexpression of enkephalin, an endogenous opioid receptor agonist, leading to opioid-dependent analgesia. Using behavioral pharmacology and single-cell RNA-seq analysis, we find that overexpression of enkephalin occurs only in cLTMR neurons, a subclass of sensory neurons involved in low-threshold touch detection, and that this overexpression does not play a role in the analgesia observed following genetic removal of Nav1.7. Furthermore, we demonstrate using laser speckle contrast imaging (LSCI) and invivo electrophysiology that Nav1.7 function is required for the initiation of C-fiber action potentials (APs), which explains the observed insensitivity to pain following genetic removal or inhibition of Nav1.7.

A novel bioassay to detect Nociceptin/Orphanin FQ release from single human polymorphonuclear cells.

Nociceptin/Orphanin FQ (N/OFQ) is the endogenous opioid agonist for the N/OFQ receptor or NOP. This receptor system is involved in pain processing but also has a role in immune regulation. Indeed, polymorphonuclear cells (PMNs) express mRNA for N/OFQ precursor and are a potential source for circulating N/OFQ. Current measurements are based on ELISA and RIA techniques. In this study we have designed a bioassay to measure N/OFQ release from single PMNs. Chinese Hamster Ovary (CHO) cells transfected with the human (h) NOP receptor and Gαiq5 chimera force receptor coupling in biosensor cells to increase intracellular Ca2+; this can be measured with FLUO-4 dye. If isolated PMNs from healthy human volunteers are layered next to CHOhNOPGαiq5 biosensor cells then stimulated with the chemoattractant N-formyl-methionyl-leucyl-phenylalanine (fMLP) we hypothesise that released N/OFQ will activate the biosensor. PMNs also release ATP and CHO cells express purinergic receptors coupled to elevated Ca2+. In a system where these receptors (P2Y1, P2Y2 and P2X7) are blocked with high concentrations of PPADS and oATP, PMN stimulation with fMLP increases Ca2+ in PMNs then shortly afterwards the biosensor cells. Our data therfore reports detection of single cell N/OFQ release from immune cells. This was absent when cells were preincubated with the selective NOP antagonist; SB-612111. Collectively this is the first description of single cell N/OFQ release. We will deploy this assay with further purified individual cell types and use this to further study the role of the N/OFQ-NOP system in disease; in particular sepsis where there is strong evidence for increased levels of N/OFQ worsening outcome.

Acute acetate administration increases endogenous opioid levels in the human brain: A [11C]carfentanil molecular imaging study.

Introduction:A recent study has shown that acetate administration leads to a fourfold increase in the transcription of proopiomelanocortin (POMC) mRNA in the hypothalamus. POMC is cleaved to peptides, including β-endorphin, an endogenous opioid (EO) agonist that binds preferentially to the µ-opioid receptor (MOR). We hypothesised that an acetate challenge would increase the levels of EO in the human brain. We have previously demonstrated that increased EO release in the human brain can be detected using positron emission tomography (PET) with the selective MOR radioligand [11C]carfentanil. We used this approach to evaluate the effects of an acute acetate challenge on EO levels in the brain of healthy human volunteers.Methods:Seven volunteers each completed a baseline [11C]carfentanil PET scan followed by an administration of sodium acetate before a second [11C]carfentanil PET scan. Dynamic PET data were acquired over 90 minutes, and corrected for attenuation, scatter and subject motion. Regional [11C] carfentanil BPND values were then calculated using the simplified reference tissue model (with the occipital grey matter as the reference region). Change in regional EO concentration was evaluated as the change in [11C]carfentanil BPND following acetate administration.Results:Following sodium acetate administration, 2.5–6.5% reductions in [11C]carfentanil regional BPND were seen, with statistical significance reached in the cerebellum, temporal lobe, orbitofrontal cortex, striatum and thalamus.Conclusions:We have demonstrated that an acute acetate challenge has the potential to increase EO release in the human brain, providing a plausible mechanism of the central effects of acetate on appetite in humans.

Naloxone regulates the differentiation of neural stem cells via a receptor-independent pathway.

The abilities of opioids to activate downstream signaling pathways normally depend on the binding between opioids and their receptors. However, opioids may also function in a receptor-independent manner, especially in neural stem cells (NSCs) in which the expression of opioid receptors and endogenous opioid agonists is low. When two opioids, morphine and naloxone, were used during the early stage of NSC differentiation, increased neurogenesis was observed. However, naloxone methiodide, a membrane impenetrable analog of naloxone, did not affect the NSC differentiation. The abilities of morphine and naloxone to facilitate neurogenesis were also observed in opioid receptor-knockout NSCs. Therefore, morphine and naloxone promote neurogenesis in a receptor-independent manner at least during the early stage. In addition, the receptor-independent functions of opioids were not observed in methylcytosinedioxygenase ten-eleventranslocation 1 (Tet1) knockout NSCs. When the expression of opioid receptors increased and the expression of Tet1 decreased during the late stage of NSC differentiation, morphine, but not naloxone, inhibited neurogenesis via traditional receptor-dependent and miR181a-Prox1-Notch-related pathway. In summary, the current results demonstrated the time-dependent effects of opioids during the differentiation of NSCs and provided additional insight on the complex functions of opioids.

The endomorphin-1/2 and dynorphin-B peptides display biased agonism at the mu opioid receptor.

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 35S-GTPγS and cAMP assays, specifically focusing on the role of adenylyl cyclases (ACs) and regulators of G-protein signaling proteins (RGSs) in CHO, N2a, and SH-SY5Y cell lines, all expressing the human MOR. We found that endomorphin-1/2 preferentially activated cAMP signaling, while dynorphin-B preferentially activated 35S-GTPγS signaling in most cell lines. Experiments carried out in the presence of an isoform selective RGS-4 inhibitor, and siRNA knockdown of AC6 in N2a cells did not significantly affect the bias properties of endomorphins, suggesting that these proteins may not play a role in endomorphin bias. 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.

Endomorphin-2 analogs containing modified tyrosines: Biological and theoretical investigation of the influence on conformation and pharmacological profile

New analogs of the endogenous opioid agonist endomorphin-2 (EM-2, H-Tyr-Pro-Phe-Phe-NH2) have been obtained by introducing modified tyrosines at the position 1 of the sequence. For all analogs, the cis/trans conformation ratio about the tyramine-Pro amide bond, lipophilicity, receptor affinities, and functional activities, have been determined. Among the novel derivatives, [Dmt(3′-Cl)]1EM-2 (4) stood out for its subnanomolar μ-opioid receptor affinity and potent agonist activity, superior to that of the parent peptide EM-2. Hybrid quantum mechanics/molecular mechanics docking computations supported the cis tyramine-Pro bioactive conformation, and allowed us to analyze the contribution of the substituents of the “message” tyramine to binding, highlighting the role of halogen-bonding in the higher receptor affinity of peptide 4.

New insights into opioid regulatory pathways: influence of opioids on Wnt1 expression in zebrafish embryos

Opioids are the most potent analgesics known today, but their prolonged administration produces severe adverse effects such as constipation, bradycardia, besides addiction, a concept not fully understood at present, which represents one of the most important challenges of modern bioscience. Wnts constitute an important family of vertebrate genes that encode secreted signaling proteins implicated in various developmental processes (patterning of the neural tube, neuronal differentiation), and are extensively conserved through evolution. In this study we have focused on Wnt1, an essential signal in axis polarity, as well as in proliferation and the development and differentiation of the CNS, roles shared by opioid receptors. Our previous studies in zebrafish show that morphine, the most potent analgesic known today, increases cell proliferation and induces neuronal protection and dopaminergic differentiation by activating the opioid receptors. The aim of the present study is to determine whether these effects are a consequence of an interaction between Wnt1 and the endogenous opioid system, which may act as a transcription regulator of Wnt1. Hence, we have exposed embryos to morphine, the endogenous delta opioid agonist Met-Enkephalin-Glu-Tyr (MEGY) (it binds with high affinity to both zebrafish delta opioid receptors, ZfDORs), and SNC80, a highly specific delta agonist, which displays low affinity towards the ZfDORs. Although at earlier stages, all opioids reduced the expression level of Wnt1, further on development, mainly during the differentiation of the CNS (24-48 h post fertilization (hpf)), morphine and MEGY increased Wnt1 expression. Our results point to the possibility that opioid signaling controls the transcription of Wnt1 and that through Wnt1, the opioid system regulates cell proliferation and neuronal differentiation. The present work opens a door to the discovery of new mechanisms that regulate opioid activity and its adverse effects, and hence, it might provide a good target to design new drugs that prevent or avoid these effects.

Opioid-substance P chimeric peptides

ABSTRACT Chimerization of tachykinin and opioid pharmacophores offers a new avenue for analgesic development. The complexities of such design are illustrated by the analgesic efficacy (via different mechanisms) of chimeras that combine pharmacophores with opioid activity and substance P activity, as well as those that combine opioid agonist and substance P antagonist moieties. Although the interaction between substance P and opioid neural systems is more complex than a simple one-way inhibition, the relative balance of activities between tachykinin and opioid pharmacophores will generally determine the net effect of the chimeric molecule as pro-nociceptive, antinociceptive, or neutral. Intriguingly, endomorphins—μ-opioid receptor agonists with high intrinsic activity—may owe some of this high activity to weak but significant antagonist proper ties at tachykinin NK2 receptors, implying that these native peptides are endogenous chimeric opioid agonist–tachykinin antagonist compounds.

The Peripheral Antinociceptive Effects of Endomorphin-1 and Kynurenic Acid in the Rat Inflamed Joint Model

Several data suggest that both opioid and N-methyl-d-aspartate (NMDA) receptors are localized at the peripheral level, and drugs acting on these receptors may produce antinociception after topical administration; however, the antinociceptive effect of endogenous ligands at these receptors is poorly clarified. Our goal in this study was to determine the antinociceptive potency of the endogenous opioid peptide, endomorphin-1 (EM1), and the endogenous NMDA receptor antagonist, kynurenic acid (KYNA), and their interaction at the peripheral level in the rat inflamed joint model. Mechanical hypersensitivity was produced by injection of carrageenan (300 microg/20 microL) into the tibiotarsal joint of the right hind leg. The mechanical pain threshold was assessed by von Frey filaments (0.064-110 g). EM1 (30, 100, and 200 microg), KYNA (30, 100, 200, and 400 microg), and their combinations in a fixed-dose ratio (1:1) were injected into the inflamed joint, and the pain threshold was determined repeatedly for 75 min after the drug administrations. Neither EM1 nor KYNA administered to the inflamed joint influenced the pain threshold at the noninflamed side. Both ligands produced dose-dependent antihyperalgesia, and the highest doses caused a prolonged effect. EM1 had higher potency (30% effective dose [ED(30)] and 50% effective dose [ED(50)] values were 112 microg [confidence interval {CI}: 80-146] and 167 microg [CI: 135-220], respectively) compared with KYNA (ED(30) and ED(50) values were 204 microg [CI: 160-251] and 330 microg [CI: 280-407], respectively). The antinociceptive effect of EM1 was prevented by subcutaneous naltrexone pretreatment. The coadministration of EM1 with KYNA caused an enhanced and/or prolonged antinociceptive effect. The ED(30) and ED(50) values of the combination were 141 microg [CI: 83-182] and 231 microg [CI: 190-293], respectively, which did not differ significantly from the theoretically additive values (ED(30) and ED(50) values were 145 microg [CI: 68-237] and 220 microg [CI: 144-230], respectively), thus the interaction between these ligands is additive. None of the treatments caused any sign of side effects. Peripherally administered endogenous opioid agonist and NMDA receptor antagonist ligands might be beneficial in inflammatory pain. Because both drugs barely cross the blood-brain barrier, their local administration causes no central side effects.

Opioids and the skin – where do we stand?

The common ectodermal origin of the skin and nervous systems can be expected to predict likely interactions in the adult. Over the last couple of decades much progress has been made to elucidate the nature of these interactions, which provide multidirectional controls between the centrally located brain and the peripherally located skin and immune system. The opioid system is an excellent example of such an interaction and there is growing evidence that opioid receptors (OR) and their endogenous opioid agonists are functional in different skin structures, including peripheral nerve fibres, keratinocytes, melanocytes, hair follicles and immune cells. Greater knowledge of these skin-associated opioid interactions will be important for the treatment of chronic and acute pain and pruritus. Topical treatment of the skin with opioid ligands is particularly attractive as they are active with few side effects, especially if they cannot cross the blood-brain barrier. Moreover, cutaneous activation of the opioid system (e.g. by peripheral nerves, cutaneous and immune cells, especially in inflamed and damaged skin) can influence cell differentiation and apoptosis, and thus may be important for the repair of damaged skin. While many of the pieces of this intriguing puzzle remain to be found, we attempt in this review to weave a thread around available data to discuss how the peripheral opioid system may impact on different key players in skin physiology and pathology.

Low doses of cyclic AMP-phosphodiesterase inhibitors rapidly evoke opioid receptor-mediated thermal hyperalgesia in naïve mice which is converted to prominent analgesia by cotreatment with ultra-low-dose naltrexone

Systemic (s.c.) injection in naïve mice of cyclic AMP-phosphodiesterase (cAMP-PDE) inhibitors, e.g. 3-isobutyl-1-methylxanthine [(IBMX) or caffeine, 10 mg/kg] or the more specific cAMP-PDE inhibitor, rolipram (1 μg/kg), rapidly evokes thermal hyperalgesia (lasting > 5 h). These effects appear to be mediated by enhanced excitatory opioid receptor signaling, as occurs during withdrawal in opioid-dependent mice. Cotreatment of these mice with ultra-low-dose naltrexone (NTX, 0.1 ng/kg–1 pg/kg, s.c.) results in prominent opioid analgesia (lasting > 4 h) even when the dose of rolipram is reduced to 1 pg/kg. Cotreatment of these cAMP-PDE inhibitors in naïve mice with an ultra-low-dose (0.1 ng/kg) of the kappa-opioid receptor antagonist, nor-binaltorphimine (nor-BNI) or the mu-opioid receptor antagonist, β-funaltrexamine (β-FNA) also results in opioid analgesia. These excitatory effects of cAMP-PDE inhibitors in naïve mice may be mediated by enhanced release of small amounts of endogenous bimodally-acting (excitatory/inhibitory) opioid agonists by neurons in nociceptive networks. Ultra-low-dose NTX, nor-BNI or β-FNA selectively antagonizes high-efficacy excitatory (hyperalgesic) Gs-coupled opioid receptor-mediated signaling in naïve mice and results in rapid conversion to inhibitory (analgesic) Gi/Go-coupled opioid receptor-mediated signaling which normally requires activation by much higher doses of opioid agonists. Cotreatment with a low subanalgesic dose of kelatorphan, an inhibitor of multiple endogenous opioid peptide-degrading enzymes, stabilizes endogenous opioid agonists released by cAMP-PDE inhibitors, resulting in conversion of the hyperalgesia to analgesia without requiring selective blockade of excitatory opioid receptor signaling. The present study provides a novel pharmacologic paradigm that may facilitate development of valuable non-narcotic clinical analgesics utilizing cotreatment with ultra-low-dose rolipram plus ultra-low-dose NTX or related agents.

Naloxone rapidly evokes endogenous kappa opioid receptor-mediated hyperalgesia in naïve mice pretreated briefly with GM1 ganglioside or in chronic morphine-dependent mice

Low-dose naloxone-precipitated withdrawal hyperalgesia is a reliable indicator of physical dependence after chronic morphine treatment. A remarkably similar long-lasting (> 3–4 h) hyperalgesia is evoked by injection of a low dose of naloxone (10 μg/kg, s.c.) in naïve mice after acute pretreatment with the glycolipid, GM1 ganglioside (1 mg/kg) (measured by warm-water-immersion tail-flick assays). GM1 treatment markedly increases the efficacy of excitatory Gs-coupled opioid receptor signaling in nociceptive neurons. Co-treatment with an ultra-low-dose (0.1 ng/kg, s.c.) of the broad-spectrum opioid receptor antagonist, naltrexone or the selective kappa opioid receptor antagonist, nor-binaltorphimine, blocks naloxone-evoked hyperalgesia in GM1-pretreated naïve mice and unmasks prominent, long-lasting (> 4 h) inhibitory opioid receptor-mediated analgesia. This unmasked analgesia can be rapidly blocked by injection after 1–2 h of a high dose of naltrexone (10 mg/kg) or nor-binaltorphimine (0.1 mg/kg). Because no exogenous opioid is administered to GM1-treated mice, we suggest that naloxone may evoke hyperalgesia by inducing release of endogenous bimodally acting opioid agonists from neurons in nociceptive networks by antagonizing putative presynaptic inhibitory opioid autoreceptors that “gate” the release of endogenous opioids. In the absence of exogenous opioids, the specific pharmacological manipulations utilized in our tail-flick assays on GM1-treated mice provide a novel bioassay to detect the release of endogenous bimodally acting (excitatory/inhibitory) opioid agonists. Because mu excitatory opioid receptor signaling is blocked by ultra-low doses of naloxone, the higher doses of naloxone that evoke hyperalgesia in GM1-treated mice cannot be mediated by activation of mu opioid receptors. Co-treatment with ultra-low-dose naltrexone or nor-binaltorphimine may selectively block signaling by endogenous GM1-sensitized excitatory kappa opioid receptors, unmasking inhibitory kappa opioid receptor signaling, and converting endogenous opioid receptor-mediated hyperalgesia to analgesia. Co-treatment with kelatorphan stabilizes putative endogenous opioid peptide agonists released by naloxone in GM1-treated mice, so that analgesia is evoked rather than hyperalgesia. Acute treatment of chronic morphine-dependent mice with ultra-low-dose naltrexone (0.1 ng/kg) results in remarkably similar rapid blocking of naloxone (10 μg/kg)-precipitated withdrawal hyperalgesia and unmasking of prominent opioid analgesia. These studies may clarify complex mechanisms underlying opioid physical dependence and opioid addiction.

Re-Discussion of the Importance of Ionic Interactions in Stabilizing Ligand-Opioid Receptor Complex and in Activating Signal Transduction

Among the many receptor classes of the GPCR family, ORs constitute a privileged drug target for their involvement in pain modulation and in a number of physiological functions and behavioural effects. Endogenous and exogenous opioid agonists have been the subject of intense investigations aiming to develop safe and potent analgesics for clinical practice; however, despite the large number of highly selective opioid agonists so far discovered, there is no convincing alternative to the use of morphine, fentanyls, and their derivatives. Alternative compounds could be very useful for treating pain forms "resistant" to the usual therapeutic agents. The recent discovery of a small number of atypical opioid agonists can furnish promising candidates for the development of alternative analgesic. In particular, a few molecules exist that can bind and activate ORs even deprived of the "minimal pharmacological requisites" generally considered to be necessary. In these cases it appears that receptor activation must be based on different ligand-receptor interaction mechanisms. Taken together, the data discussed in the review suggest that the prevailing assumptions about OR binding need revision. In particular, they strengthen the evidence that ORs can bind ligands via diverse binding modes, and in some cases an electrostatic interaction is not an absolute requirement.

P21 Therapeutic Efficacy of Plasmapheresis for the Treatment of Refractory Pruritis of Primary Billiary Cirrhosis: 2 Case Reports.

Primary billiary cirrhosis (PBC), a chronic cholestatic disease of presumed autoimmune aetiology, is characterised by progressive intrahepatic bile duct destruction, liver cirrhosis, failure, and death, unless treated by liver transplantation. Distressing pruritis (skin itch), fatigue and metabolic bone diseases frequently complicate this disease, significantly reducing quality of life. The pathogenesis of pruritis remains uncertain. Some unknown pruritogen(s) produced by the liver have been implicated which accumulate in the plasma as a result of chronic cholestasis. Recent evidence also suggests a central mechanism, with increased neurotransmission/neuromodulation mediated by endogenous opioid agonists. Therapeutic interventions include unabsorbable oral resins, urso‐deoxycholeic acid, opiate antagonists and rifampicin. More experimental therapies include: partial diversion of bile; charcoal haemoperfusion; UVA; plasmapheresis and albumin‐based dialysis (for patients with renal impairment). We used plasma exchange for the treatment of intractable pruritis in two patients with primary billiary cirrhosis. Both were females between ages of 50–60 years, refractory to all medical treatments and severely disabled by intractable pruritis and fatigue. In the first patient, weekly to fortnightly plasmapheresis considerably reduced the intensity of itch and stabilized her condition until her liver transplant. The second patient was treated for a relapse of PBC following her liver transplant. She significantly improved with more than 50% reduction in the intensity of her generalized itch with healing of excoriations, and was able to sleep for 4 h undisturbed after only one procedure. We suggest that plasmapheresis may be helpful for the pruritis of chronic cholestasis refractory to medical management. It could also be considered for the initial treatment of selected patients in combination with other therapies.

75 years of opioid research: the exciting but vain quest for the Holy Grail

Over the 75-year lifetime of the British Pharmacological Society there has been an enormous expansion in our understanding of how opioid drugs act on the nervous system, with much of this effort aimed at developing powerful analgesic drugs devoid of the side effects associated with morphine--the Holy Grail of opioid research. At the molecular and cellular level multiple opioid receptors have been cloned and characterised, their potential for oligomerisation determined, a large family of endogenous opioid agonists has been discovered, multiple second messengers identified and our understanding of the adaptive changes to prolonged exposure to opioid drugs (tolerance and physical dependence) enhanced. In addition, we now have greater understanding of the processes by which opioids produce the euphoria that gives rise to the intense craving for these drugs in opioid addicts. In this article, we review the historical pathway of opioid research that has led to our current state of knowledge.

Role of Endogenous Opioid Receptor Agonists in Regulation of Heart Resistence to the Arrhythmogenic Action of Short-Term Ischemia and Reperfusion

Preliminary selective block of mu-, delta1-, delta2-, and kappa-opioid receptors had no effect on the incidence of ventricular arrhythmias during 10-min coronary occlusion-reperfusion in ketamine-narcotized rats. Repetitive short-term immobilization of rats for 2 weeks improved heart resistance to the arrhythmogenic action of coronary occlusion and reperfusion. Selective mu-opioid receptor antagonist CTAP completely abolished, while selective delta- and kappa-opioid receptor antagonists did not modulate the antiarrhythmic effect of adaptation. Probably, endogenous agonists of mu-opioid receptors play an important role in the adaptive improvement of heart resistance to arrhythmogenic factors, but are insignificant for the modulation of heart resistance to the arrhythmogenic action of short-term local ischemia-reperfusion in non-adapted animals.

Modulation of Synaptic Transmission in the Rat Nucleus of the Solitary Tract by Endomorphin-1

Activation of opioid receptors in the periphery and centrally in the brain results in inhibition of gastric and other vagally mediated functions. The aim of this study was to examine the role of the endogenous opioid agonist endomorphin 1 (EM-1) in regulating synaptic transmission within the nucleus tractus solitarius (NTS), an integration site for autonomic functions. We performed whole cell patch-clamp recordings from coronal brain slices of the rat medulla. A subset of the neurons studied was prelabeled with a stomach injection of the transsynaptic retrograde virus expressing EGFP, PRV-152. Solitary tract stimulation resulted in constant latency excitatory postsynaptic currents (EPSCs) that were decreased in amplitude by EM-1 (0.01-10 microM). The paired-pulse ratio was increased with little change in input resistance, suggesting a presynaptic mechanism. Spontaneous EPSCs were decreased in both frequency and amplitude by EM-1, and miniature EPSCs were reduced in frequency but not amplitude, suggesting a presynaptic mechanism for the effect. Spontaneous inhibitory postsynaptic currents (IPSCs) were also reduced in frequency by EM-1, but the effect was blocked by TTX, suggesting activity at receptors on the somata of local inhibitory neurons. Synaptic input arising from local NTS neurons, which were activated by focal photolysis of caged glutamate, was inhibited by EM-1. The actions of EM-1 were similar to those of D-Ala2, N-Me-Phe4, Gly5-ol]-enkephalin (DAMGO) and were blocked by naltrexone, D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2 (CTOP), or D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2 (CTAP). These results suggest that EM-1 acts at mu-opioid receptors to modulate viscerosensory input and specific components of local synaptic circuitry in the NTS.

Peripheral effect of a kappa opioid receptor antagonist on nociception evoked by formalin injected in TMJ of pregnant rats

The effect of sex hormones on orofacial pain modulation is poorly understood. Therefore, this study aimed to investigate the effect of hormonal changes as a result of pregnancy, as well as that of the kappa (κ) opioid receptor antagonist on female rats' sensitivity to the temporomandibular joint (TMJ) formalin test. Initially, female rats at estrus and pregnant females on day 19 of pregnancy received a 50 μl formalin (1.5%) injection in the right TMJ. The pregnant females showed a reduction in nociceptive responses to the TMJ formalin test when compared with those at estrus. Then, the selective κ-opioid receptor antagonist nor-Binaltorphimine (nor-BNI), was co-administered with the formalin. Next, additional groups received the κ (200 μg) receptor antagonist or 0.9% NaCl 24 hours prior to the periarticular injection of formalin. Co-administration of nor-BNI with formalin into the TMJ region had no significant effect. The pre-injection of selective κ-opioid receptor antagonist, nor-BNI, significantly enhanced the nociceptive behavioral responses in pregnant females. When applied in the contralateral TMJ, nor-BNI did not affect the magnitude of the nociceptive response induced by formalin. It can be concluded that: 1) The increase of the sex hormone levels, as result of pregnancy, induces a reduction of nociceptive behavioral responses to the TMJ formalin test; 2) the peripheral kappa opioid receptor activation, by endogenous opioid agonists release, is involved in the antinociception to TMJ formalin test, induced by pregnancy.

Selective Postsynaptic Inhibition of Tonic-firing Neurons in Substantia Gelatinosa by μ-Opioid Agonist

Spinal substantia gelatinosa (SG) is a site of action of administered and endogenous opioid agonists and is an important element in the system of antinociception. However, little is known about the types of neurons serving as specific postsynaptic targets for opioid action within the SG. To study the spinal mechanisms of opioidergic analgesia, the authors compared the action of mu-opioid agonist [D-Ala, N-Me-Phe, Gly-ol]-enkephalin (DAMGO) on SG neurons with different intrinsic firing properties. Whole cell patch clamp recordings from spinal cord slices of Wistar rats were used to study the sensitivity of SG neurons to DAMGO. Three groups of neurons with distinct distributions in SG were classified: tonic-, adapting-, and delayed-firing neurons. DAMGO at 1 microm concentration selectively hyperpolarized all tonic-firing neurons tested, whereas none of the adapting- or delayed-firing neurons were affected. The effect of DAMGO on tonic-firing neurons was due to activation of G protein-coupled inward-rectifier K conductance, which could be blocked by 500 microm Ba and 500 microm Cs but increased by 50 microm baclofen. As a functional consequence of DAMGO action, a majority of tonic-firing neurons changed their pattern of intrinsic firing from tonic to adapting. It is suggested that tonic-firing neurons, presumably functioning as excitatory interneurons, are primary postsynaptic targets for administered and endogenous opioid agonists in spinal SG. Functional transition of cells in this group from tonic to adapting firing mode may represent an important mechanism facilitating opioidergic analgesia.