Non-selective Opioid Antagonist Naloxone Research Articles

Suboxone: History, controversy, and open questions.

There are more than 200 opioid overdose deaths each day in the US. In combating this epidemic we look to available treatment tools. Here, we find only three medications approved by the Food and Drug Administration (FDA) for the treatment of opioid use disorder. Of the three, buprenorphine is of particular importance due to its reduced overdose potential as a partial opioid agonist. Evidence supports its clinical equivalence to its full agonist cousin methadone, and suggests that it is better slated for long-term treatment of opioid use disorder compared to the non-selective opioid antagonist naltrexone. Buprenorphine is most popularized within Suboxone, a medication which also contains the non-selective opioid antagonist naloxone. The naloxone has no additional effect when the drug is taken as instructed, as it is intended to prevent diversion in those that would attempt to inject the medication. While Suboxone is regarded by some as the future of medical treatment, others have expressed concerns. This review aims to explore the history, controversy, and open questions that surround buprenorphine and its most prescribed variation, Suboxone. These include its pharmacological, legislative, and social history, alternative indications, efficacy as a treatment of opioid use disorder, and more. Armed with this information, the reader will have a more in-depth and holistic understanding of the medication's place in their community.

Supraspinal melatonin MT2 receptor agonism alleviates pain via a neural circuit that recruits mu opioid receptors.

Melatonin, through its G protein-coupled receptor (GPCR) (MTNR1B gene)MT2 , is implicated in analgesia, but the relationship between MT2 receptors and the opioid system remains elusive. In a model of rodent neuropathic pain (spared nerve injured [SNI]), the selective melatonin MT2 agonist UCM924 reversed the allodynia (a pain response to a non-noxious stimulus), and this effect was nullified by the pharmacological blockade or genetic inactivation of the mu opioid receptor (MOR), but not the delta opioid receptor (DOR). Indeed, SNI MOR, but not DOR knockout mice, did not respond to the antiallodynic effects of the UCM924. Similarly, the nonselective opioid antagonist naloxone and the selective MOR antagonist D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2 (CTOP) blocked the effects of UCM924 in SNI rats, but not the DOR antagonist naltrindole (NTI). Electrophysiological recordings in the rostral-ventromedial medulla (RVM) revealed that the typical reduction of the firing activity of pronociceptive ON-cells, and the enhancement of the firing of the antinociceptive OFF-cells, induced by the microinjection of the MT2 agonist UCM924 into the ventrolateral periaqueductal gray (vlPAG) were blocked by MOR, but not DOR, antagonism. Immunohistochemistry studies showed that MT2 receptors are expressed in both excitatory (CaMKIIα+ ) and inhibitory (GAD65+ ) neuronal cell bodies in the vlPAG (~2.16% total), but not RVM. Only 0.20% of vlPAG neurons coexpressed MOR and MT2 receptors. Finally, UCM924 treatment induced an increase in the enkephalin precursor gene (PENK) in the PAG of SNI mice. Collectively, the melatonin MT2 receptor agonism requires MORs to exert its antiallodynic effects, mostly through an interneuronal circuit involving MOR and MT2 receptors.

Quantification of kappa opioid receptor ligand potency, efficacy and desensitization using a real-time membrane potential assay

We explored the utility of the real-time FLIPR Membrane Potential (FMP) assay as a method to assess kappa opioid receptor (KOR)-induced hyperpolarization. The FMP Blue dye was used to measure fluorescent signals reflecting changes in membrane potential in KOR expressing CHO (CHO-KOR) cells. Treatment of CHO-KOR cells with kappa agonists U50,488 or dynorphin [Dyn (1−13)NH2] produced rapid and concentration-dependent decreases in FMP Blue fluorescence reflecting membrane hyperpolarization. Both the nonselective opioid antagonist naloxone and the κ-selective antagonists nor-binaltorphimine (nor-BNI) and zyklophin produced rightward shifts in the U50,488 concentration-response curves, consistent with competitive antagonism of the KOR mediated response. The decrease in fluorescent emission produced by U50,488 was blocked by overnight pertussis toxin pretreatment, indicating the requirement for PTX-sensitive G proteins in the KOR mediated response. We directly compared the potency of U50,488 and Dyn (1−13)NH2 in the FMP and [35S]GTPγS binding assays, and found that both were approximately 10 times more potent in the cellular fluorescence assay. The maximum responses of both U50,488 and Dyn (1−13)NH2 declined following repeated additions, reflecting receptor desensitization. We assessed the efficacy and potency of structurally distinct KOR small molecule and peptide ligands. The FMP assay reliably detected both partial agonists and stereoselectivity. Using KOR-selective peptides with varying efficacies, we found that the FMP assay allowed high throughput quantification of peptide efficacy. These data demonstrate that the FMP assay is a sensitive method for assessing κ-opioid receptor induced hyperpolarization, and represents a useful approach for quantification of potency, efficacy and desensitization of KOR ligands.

Endogenous Opioid Receptor Activation in the Caudal Medullary Raphe Depresses Respiratory Rate in Decerebrate Rabbits

Background The caudal medullary raphe (CMR) is an essential structure in controlling pain, cardiovascular, and respiratory function (1). Partial reversal of systemic opioid-induced respiratory depression was achieved with naloxone injection into the CMR (2). This study investigated the presence of endogenous opioid activity in the CMR. Methods The study was approved by the local Animal Care Committee and conformed to NIH standards. Adult New Zealand White rabbits (3-4kg) were anesthetized, tracheotomized, ventilated (FiO2 0.6, PCO2 45-55 mmHg), decerebrated and vagotomized. Phrenic nerve activity was recorded from the c5 rootlet, time averaged and used to calculate inspiratory (TI) and expiratory (TE) duration, breaths per minute (BPM), and peak phrenic activity (PPA). Using a multibarrel glass micropipette, we performed the following injection protocols into the CMR in the midline between 2 mm caudal and 1.5 mm rostral to the functionally identified preBötzinger Complex (preBC, (3)) and into the middle medullary raphe (middle MR (2), 2.5-3.5mm rostral to the preBC) in separate sets of animals: i) α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA, 50 μM, 70 nl), stepsize 0.5mm rostro-caudal, 0.5mm dorso-ventral. ii) The non-selective opioid antagonist naloxone (1-5 mM, 700 nl), stepsize 0.5mm rostro-caudal, 1st injection at the ventral edge of neuronal activity, 2nd injection 1.5-2mm dorsal; and iii) the µ-opioid antagonist CTAP and δ-opioid antagonist naltrindole (each 1 mM, 350-700 nl), stepsize same as ii). Statistical comparison with Wilcoxon signed rank test. Median (25-75% range). Results i) In 6 animals, AMPA injection into the CMR caudal to and at the level of the preBC increased respiratory rate by up to 59% and PPA by up to 38% at multiple dorso-ventral levels. This was due to a decrease in TI and TE. In the middle MR, AMPA injection depressed respiratory rate, at times to apnea, through an increase in TI and TE, and also depressed PPA. ii) Naloxone injection into the CMR increased respiratory rate from 30 (23-34) to 37 (30-6) BPM (p=0.031, n=6) through a decrease in TI from 0.9 (0.8-1.0) to 0.7 (0.6-0.8) sec (p=0.031) and TE from 1.2 (1.0-1.6) to 0.9 (0.7-1.2) sec (p=0.031). We observed effects with injections caudal to, at the level of and rostral to the preBC. iii) CTAP injection increased respiratory rate from 26 (22-27) to 34 (29-34) BPM, and subsequent naltrindole injection increased respiratory rate to 43 (37-44) BPM (n=3). In the middle MR, CTAP + naltrindole increased respiratory rate between 2-5 BPM. Conclusion Excitation of the CMR increased respiratory rate over an area of several millimeters. During quiet breathing under moderate hyperoxic hypercapnia in our decerebrate rabbit preparation, respiratory raphe neurons were depressed by endogenous opioid activity. It will be important to further investigate the role of the CMR in opioid-induced respiratory depression. (1) Holtman et al., J Neurosci 1986, 6: 1185-93 (2) Zhang et al., Anesthesiology 2007, 107: 288-97 (3) Cook-Snyder et al., Resp Physiol Neurobiol 2019, 260: 37-52

Beta-endorphin inhibits the inflammatory response of bovine endometrial cells through δ opioid receptor in vitro

Postpartum uterine infections are common reproductive diseases in postpartum cows. Evidence has shown that plasma β-endorphins increase during bovine uterine inflammation. However, the effect of β-endorphins on the inflammatory response in bovine endometrium has not been clarified. The aim of this study was to investigate the effect of β-endorphins on the inflammatory response of bovine endometrial epithelial and stromal cells, and to explore the possible mechanism. The cells were treated with E. coli lipopolysaccharide (LPS) to simulate inflammation, which was characterized by the significant activation of NF-κB signaling pathway and the increased gene expression of the downstream proinflammatory cytokines (approximately 1.2- to 15-fold increase, P < 0.05). By using Western blot and qPCR techniques, we found that β-endorphins inhibited the key protein expression of NF-κB pathway, and the gene expressions of TNF, IL1B, IL6, CXCL8, nitric oxide synthase 2, and prostaglandin-endoperoxide synthase 2 (P < 0.05). The co-treatment of β-endorphins and opioid antagonists showed that the anti-inflammatory effect of β-endorphins could be blocked (P < 0.05) by non-selective opioid antagonist naloxone or δ opioid receptor antagonist ICI 154129, but not the μ opioid receptor antagonist CTAP (P > 0.05). In conclusion, β-endorphins may inhibit the inflammatory response of bovine endometrial epithelial and stromal cells through δ opioid receptor.

In vivo Dependence Liability Assessment of Novel Fentanyl Analogues: Science to Guide drug Policy

The emergence of fentanyl analogues as drugs of abuse raises concern regarding their dependence liability. Following prolonged opioid administration, physical dependence leads to increased use, which facilitates the development of tolerance and the dangers of overdose. In animal models, opioid dependence can be inferred by assessing signs of withdrawal, with vertical jumping being the most sensitive and easily quantified in the mouse. In this study, we aimed to characterize the dependence liability of fentanyl, morphine, and six fentanyl analogues by assessing antinociception and withdrawal signs elicited by each compound in the mouse. First, to determine antinociceptive effects, we used a warm‐water tail withdrawal procedure using both 50°C and 55°C water. Latencies until mice removed their tails from the water were recorded, and fully effective doses were determined using a 10 second cut‐off to prevent tissue damage. Dose effect‐curves were generated to determine the potency (ED50) of each compound for eliciting antinociception. All analogues were less potent than fentanyl but more potent than morphine. Additionally, all test compounds were fully effective in both 50°C and 55°C water. Next, to assess withdrawal, we treated animals with 3 injections of morphine, fentanyl or the various fentanyl analogues spaced 12 hours apart. Following the first two injections on day 1, we precipitated withdrawal on day 2, 3 hours after the final injection of agonist, using the nonselective opioid antagonist naloxone. Withdrawal sessions lasted 20 minutes and the number of vertical jumps were recorded for each session. Antagonist dose‐effect curves for withdrawal jumps were generated first using an agonist dose one‐log unit higher than the fully effective antinociceptive dose and precipitating withdrawal with 3, 10, and 30 mg/kg of naloxone. Next, we used 30 mg/kg of naloxone to precipitate withdrawal using increasing unit doses of each test compoewund since maximal jumping was elicited using 30 mg/kg of naloxone. Agonist dose‐effect curves were generated starting with the lowest fully effective antinociceptive dose and increasing in half‐log units. Our results indicate that all of these fentanyl derivatives are capable of eliciting dependence and offer valuable insight into their abuse potential.Support or Funding InformationThese studies supported in part by DEA/NCTR (224‐16‐0505R),) and the University of Arkansas for Medical ScienVA (D‐15‐OD‐002cesThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Opioidergic effects of transcutaneous electrical nerve stimulation on pain and inflammatory edema in a rat model of ankle sprain

ABSTRACT Although transcutaneous electrical nerve stimulation (TENS) has been proposed to modulate pain and the mechanisms underlying analgesia remain poorly understood, evidence of anti-inflammatory effect is more limited. The purpose of this study was to examine the opioidergic mechanisms of TENS effects in two different frequencies on pain and inflammatory edema in the ankle sprain model in rats. Threshold to mechanical stimulation was utilized to examine the changes produced by intraperitoneal injection of non-selective opioid antagonist naloxone on the antihyperalgesic effect induced by a 20-min period of 2Hz or 100Hz TENS in the ankle sprain model, produced by manually overextending the lateral ligaments. Ankle sprain induced a long-lasting reduction in paw withdrawn latency (PWL) after 30 minutes for up to 24 hours in sham TENS (SH-TENS) treated rats. The reduced PWL after the induction of ankle sprain was restored partially at 0,1,2,3 and 6, but not 24 hours, after the termination of 2 Hz-TENS (LF-TENS). In 100Hz (HF-TENS) the reduction in PWL was shorter than LF-TENS and both LF and HF effects were fully blocked in naloxone-treated rats. LF- and HF-TENS treated rats did not reach the elevation of edema and presented a progressive edema reduction for over 24 hours when compared to SH-TENS group. Both effects were reduced by naloxone. TENS-induced antihyperalgesic and anti-edematous effects observed in ankle sprain model were mediated by the endogenous opioid system.

Activation of κ Opioid Receptors in Cutaneous Nerve Endings by Conorphin-1, a Novel Subtype-Selective Conopeptide, Does Not Mediate Peripheral Analgesia.

Selective activation of peripheral κ opioid receptors (KORs) may overcome the dose-limiting adverse effects of conventional opioid analgesics. We recently developed a vicinal disulfide-stabilized class of peptides with subnanomolar potency at the KOR. The aim of this study was to assess the analgesic effects of one of these peptides, named conorphin-1, in comparison with the prototypical KOR-selective small molecule agonist U-50488, in several rodent pain models. Surprisingly, neither conorphin-1 nor U-50488 were analgesic when delivered peripherally by intraplantar injection at local concentrations expected to fully activate the KOR at cutaneous nerve endings. While U-50488 was analgesic when delivered at high local concentrations, this effect could not be reversed by coadministration with the selective KOR antagonist ML190 or the nonselective opioid antagonist naloxone. Instead, U-50488 likely mediated its peripheral analgesic effect through nonselective inhibition of voltage-gated sodium channels, including peripheral sensory neuron isoforms NaV1.8 and NaV1.7. Our study suggests that targeting the KOR in peripheral sensory nerve endings innervating the skin is not an alternative analgesic approach.

κ-opioid receptors in the infralimbic cortex modulate the cardiovascular responses to acute stress.

What is the central question of this study? A brief experience of stress can cause structural remodelling in the infralimbic cortex. In the present study, we addressed the potential role played by opioidergic neurotransmission in the infralimbic cortex in the modulation of stress-evoked autonomic responses. What is the main finding and its importance? Using the restraint stress model, we showed that infralimbic cortex κ-opioid receptors, but not μ- and δ-opioid receptors, modulate stress-evoked cardiovascular responses. The infralimbic cortex (IL) is known to modulate behavioural and physiological responses during aversive situations. We investigated the hypothesis that opioid neurotransmission in the IL modulates the autonomic responses induced in rats subjected to restraint stress (RS). Male Wistar rats (250-280 g) were used. Guide cannulae were implanted bilaterally in the IL for the microinjection of either drugs or vehicle, and a polyethylene catheter was implanted into the femoral artery for recording of mean arterial pressure (MAP) and heart rate (HR) using a computerized acquisition system. Tail temperature was evaluated using a thermal camera. Rats were subjected to RS 10 min after the microinjection of drugs or vehicle into the IL. Exposure to RS evoked hypertension, tachycardia and a reduction in tail temperature. Bilateral microinjections of the non-selective opioid antagonist naloxone into the IL generated an inverted U-shaped dose-inhibition curve on RS-evoked MAP and HR responses. Microinjection of nor-BNI (κ-selective antagonist) reduced the increases in MAP and HR evoked by RS. In contrast, pretreatment of the IL with CTAP (μ-selective antagonist) or naltrindole (δ-selective antagonist) had no effect on the restraint-evoked increases in MAP and HR. None of these treatments altered the reduction in the tail temperature evoked by RS. In conclusion, κ-opioid receptors in the IL modulate pressor and tachycardiac responses caused by RS, suggesting a facilitatory role of this structure in this aversive situation.

Opioid receptors in the prelimbic cortex modulate restraint stress-induced cardiovascular responses in the rat

The prelimbic cortex (PL) is involved in the control of behavioral and autonomic responses to stress. The present study aimed to investigate whether opioid neurotransmission in the PL modulates autonomic responses evoked by restraint stress (RS). Bilateral microinjection of 0.03, 0.3 and 3 nmol/100 nL of the nonselective opioid antagonist naloxone into the PL reduced pressure and tachycardiac responses evoked by RS. However, no effects were observed after its injection at doses of 0.003 and 30 nmol/100 nL, thus resulting in an inverted U-shaped dose–inhibition curve. Similar to naloxone, the selective μ-opioid antagonist CTAP, and the selective κ-opioid antagonist nor-BNI, also reduced MAP and HR increases induced by RS when injected into the PL, whereas treatment with the selective δ-opioid antagonist naltrindole did not affect the pressor and tachycardiac response caused by RS. Blockade of opioid neurotransmission in the PL did not affect the fall in tail temperature and increase in body temperature induced by RS. The present results confirm the involvement of PL opioid neurotransmission in the modulation of cardiovascular responses evoked during the exposure to an aversive situation, and suggest that responses observed after the blockade of local opioid receptors is due to alterations in PL neuronal activity. Furthermore, these results suggest that a distinct circuitry is involved in modulation of the sympathetic output to different vascular territories.

Correlates of recovery from incentive downshift: A preliminary selective breeding study

Rats exposed to a downshift in the concentration of a sucrose solution from 32% to 4% exhibit a transient suppression of consummatory behavior relative to an unshifted control always exposed to 4% sucrose. One explanation of this effect, known as consummatory successive negative contrast (cSNC), explains consummatory suppression as arising from an emotional state of frustration that redirects behavior away from the source of the devalued solution. A preliminary selective breeding protocol consisting of three experiments was performed. Experiment 1 reports results from 5 generations of selected breeding for either high (H) or low (L) recovery rates from cSNC. A control line of randomly (R) mated rats was included. cSNC was reduced in H rats, but L and R rats did not differ across generations. H rats also provided no evidence of behavioral activation in acquisition or increased persistence in extinction after partial reinforcement, rather than continuous reinforcement. L and R rats, by contrast, showed both of these effects. H rats were also significantly smaller in body size than R rats, but did not differ in terms of water intake, sucrose sensitivity, open-field activity, or responding to sucrose solutions before the downshift. In Experiment 2, H infants from the sixth selected generation showed increased bandwidth in vocalizations induced by mother-infant separation relative to L and R rats. Experiment 3 showed that H rats failed to show increased response to incentive downshift after treatment with the nonselective opioid antagonist naloxone, as done by L and R rats. The results, if replicated, may provide support for the interpretation of a significant role of frustration in cSNC.

Endogenous opioids regulate alveolar bone loss in a periodontal disease model

AimThe anti-inflammatory effects of exogenous opioid compounds have been demonstrated in several conditions. Nevertheless, the function of endogenous opioid peptides released by the host during inflammatory processes deserves further characterization. The aim of this study was to verify whether endogenous opioids are involved in the progression of the inflammatory alveolar bone loss induced by ligature in rats. Main methodsThe experimental model of periodontal disease (PD) induced by ligature in rats was used throughout the study. A silk ligature was placed around the 2nd upper molar of male Holtzman rats, for 7days. Rats received different doses of either the non-selective opioid antagonist naloxone or vehicle, locally into the afflicted gingival tissue, from the 3rd to the 5th day after ligature placement. In the 7th experimental day, rats were euthanized and their maxillae were collected for evaluation of alveolar bone and fiber attachment loss, presence of neutrophils (myeloperoxidase assay), osteoclast amount, and levels of cytokines IL-6, TNF-α, IL-8 and IL-10 in periodontal tissues. Key findingsNaloxone increased alveolar bone loss significantly, in a dose-dependent manner, in relation to vehicle-treated rats. In contrast, the opioid antagonist did not affect the loss of fiber attachment. The treatment with naloxone also induced a significant increase in myeloperoxidase levels, osteoclast number and cytokines in periodontal tissues of rats with ligature-induced PD. SignificanceEndogenous opioids protect the host from the progression of inflammatory alveolar bone loss that occurs in chronic periodontitis.

Paroxetine Augments while Naloxone Abolishes the Analgesic Effect of Paracetamol in Acute Nociceptive Pain in Mice

The mechanism(s) of analgesic action of paracetamol (acetaminophen; N-acetyl-p-aminophenol) remains controversial. Previous studies on rats suggested that the antinociceptive action of paracetamol might involve the central descending inhibitory pain pathways recruiting both a serotoninergic and an opioidergic system. This study explores this issue in mice using paroxetine, the most potent selective serotonin re-uptake inhibitor, and the nonselective opioid pure antagonist naloxone. Animals were divided into two main groups for two separate experiments, each subdivided into 3 subgroups. In both experiments; the first group served as control, the second group received paracetamol (200 mg/kg, i.p). In one experiment, the third group received paroxetine (20 mg/kg p.o for 7 days) before paracetamol. In the other experiment, animals of the third group were pretreated with naloxone (5 mg/kg, i.p) 30 min before paracetamol. The antinociceptive effect of paracetamol was tested using the hot plate test. Paracetamol displayed a significant antinociceptive activity that was augmented by pretreatment with paroxetine as was shown by maintenance of its effect beyond that shown by paracetamol alone. On the other hand, pretreatment with naloxone abolished paracetamol’s antinociceptive activity in the hot-plate test. These results extended the previous observation in rats that the antinociceptive effect of paracetamol involved activation of a central descending pain inhibitory pathway with serotonin and opioidergic peptides being potential mediators recruited.

Opioid‐like antinociceptive effects of oral administration of a lectin purified from the seeds of Canavalia brasiliensis

The objective of this study was to evaluate the antinociceptive effects of a lectin from Canavalia brasiliensis (ConBr) when administered orally to murine models of chemical and thermal nociception. ConBr up to 100 mg/kg produced significant and dose-dependent antinociceptive effects: 81% reduction in abdominal writhing induced by 0.6% acetic acid; 26 and 52% reduction in early- and late-stage paw licking, respectively, induced by 2.5% formalin; and 155% increase in reaction latency (heightened thermal pain threshold). In all models, the antinociceptive effect was reversed by the lectin-binding carbohydrate α-d-methyl-mannoside and by the nonselective opioid antagonist naloxone. The antinociceptive effect observed in the formalin test was inhibited by the δ-selective antagonist naltrindole and the κ-selective antagonist nor-binaltorphimine but not by the μ-selective antagonist cyprodime. In conclusion, when administered orally to Swiss mice, the ConBr lectin displayed antinociceptive activity, both peripheral and central, mediated by the opioid system and involving δ-and κ-receptors and the lectin domain.

The action of a synthetic derivative of Met5-enkephalin-Arg6-Phe7 on behavioral and endocrine responses

The neuroendocrine and behavioral effects of Tyr-d-Ala-Gly-Phe-d-Nle-Arg-Phe (DADN), a more stable derivative of the endogenous opiate Met-enkephalin related peptide Met5-enkephalin-Arg6-Phe7 were investigated in mice. The behavioral experiments consisted of monitoring the horizontal (square crossing) and vertical (rearing) locomotion in the open field system. To evaluate the effect of the heptapeptide on the hypothalamo–pituitary–adrenal (HPA) axis, the plasma corticosterone level was measured. DADN induced dose-dependent increases in locomotion and rearing 30min after intracerebroventricular injection and also elicited marked activation of the hormonal stress response. To elucidate the receptors involved in the mediation of these actions, animals were pretreated with the nonselective opioid antagonist naloxone, the selective κ-receptor antagonist nor-binaltorphimine or the μ1-receptor blocker naloxonazine. Both the HPA activation and the behavioral responses were diminished by the preadministration of naloxone. Nor-binaltorphimine did not display a significant effect, while naloxonazine completely abolished the hyperactivity and the corticosterone elevation elicited by the analog. These findings suggest that μ-receptors predominate in the mediation of the neuroendocrine actions of DADN, while κ-receptors do not play a significant role.

κ-Opioid Receptor Inhibition of Calcium Oscillations in Spinal Cord Neurons

Mouse embryonic spinal cord neurons in culture exhibit spontaneous calcium oscillations from day in vitro (DIV) 6 through DIV 10. Such spontaneous activity in developing spinal cord contributes to maturation of synapses and development of pattern-generating circuits. Here we demonstrate that these calcium oscillations are regulated by κ opioid receptors (KORs). The κ opioid agonist dynorphin (Dyn)-A (1-13) suppressed calcium oscillations in a concentration-dependent manner, and both the nonselective opioid antagonist naloxone and the κ-selective blocker norbinaltorphimine eliminated this effect. The KOR-selective agonist (+)-(5α,7α,8β)-N-methyl-N-[7-(1-pyrrolidinyl)-1-oxaspiro[4.5]dec-8-yl]-benzeneacetamide (U69593) mimicked the effect of Dyn-A (1-13) on calcium oscillations. A κ-specific peptide antagonist, zyklophin, was also able to prevent the suppression of calcium oscillations caused by Dyn-A (1-13). These spontaneous calcium oscillations were blocked by 1 μM tetrodotoxin, indicating that they are action potential-dependent. Although the L-type voltage-gated calcium channel blocker nifedipine did not suppress calcium oscillations, the N-type calcium channel blocker ω-conotoxin inhibited this spontaneous response. Blockers of ionotropic glutamate receptors, 2,3-dihydroxy-6-nitro-7-sulfamoylbenzo(f)quinoxaline and dizocilpine maleate (MK-801), also suppressed calcium oscillations, revealing a dependence on glutamate-mediated signaling. Finally, we have demonstrated expression of KORs in glutamatergic spinal neurons and localization in a presynaptic compartment, consistent with previous reports of KOR-mediated inhibition of glutamate release. The KOR-mediated inhibition of spontaneous calcium oscillations may therefore be a consequence of presynaptic inhibition of glutamate release.

Ethanol‐Induced Social Facilitation in Adolescent Rats: Role of Endogenous Activity at Mu Opioid Receptors

Ethanol consumption is considerably elevated during adolescence. Attractiveness of alcohol for humans during the adolescent developmental period is based, in part, on its ability to induce social facilitation--a facilitation of social interactions not only evident in human adolescents but also in adolescent rats. Endogenous opioid systems are among the multiple neural systems implicated in the behavioral and reinforcing effects of ethanol and may play a substantial role in modulating stimulatory effects of low doses of ethanol on social behavior during adolescence. This possibility was explored in the present study through the use of an animal model of peer-directed social behavior. Sprague-Dawley rats were challenged early in adolescence with saline or ethanol intraperitoneally (i.p.), placed into an individual holding cage for 30 minutes, and then tested in a familiar situation with a nonmanipulated partner of the same age and sex. In Experiment 1, each test subject was injected subcutaneously with one of the three doses of a nonselective opioid antagonist naloxone (0, 0.05, and 0.1 mg/kg), 5 minutes prior to the social interaction test and 25 minutes following challenge with saline or ethanol (0.5 g/kg), whereas in Experiment 2 animals were challenged with one of the six doses of ethanol (0, 0.25, 0.5, 0.75, 1.0, and 1.25 g/kg) prior to injection of either saline or naloxone (0.05 mg/kg). In Experiment 3, animals were pretreated i.p. with the selective mu-opioid antagonist CTOP (0, 0.01, 0.025, 0.05, and 0.1 mg/kg) 30 minutes prior to challenge with saline or ethanol (0.5 g/kg). Low doses of ethanol (0.5 and 0.75 g/kg) produced social facilitation, as indexed by significant increases in play fighting and social investigation. Both doses of naloxone and the three highest doses of CTOP blocked the stimulatory effects of ethanol on play fighting but not on social investigation. These effects were not associated with alterations in ethanol pharmacokinetic properties or with shifts in the biphasic ethanol dose-response curve. Ethanol-induced facilitation of social play behavior seen in adolescent animals is mediated in part through ethanol-induced release of endogenous ligands for the mu-opioid receptor or an ethanol-associated enhancement of sensitivity of these receptors for their endogenous ligands.

604 H 2S Induces Analgesia Is Mediated In Vivo Activation of μ Opioid Receptor and PI3K/AKT Pathway Activation

Background and aim. Hydrogen sulfide (H2S) functions as a neuromodulator and reverses colorectal distension (CRD)-induced pain. Opioid system is widely involved in analgesia. Whether the opioid receptors are involved in the H2S -induced analgesia is unknown. Methods. Four, graded (0.4-1.6 ml water) CRD were produced in awake rats and colorectal pain were assessed by measuring the abdominal withdrawal response (AWR) after i.p. administration of saline or Na2S, a donor of H2S (100 mMol/kg). Spinal cFos expression was also detected. To determine whether the opioid system was involved in the H2S -induced antinociception, pre-treatment with non-selective opioid antagonist naloxone (0.5 mg/kg i.p.) or selective antagonists of μ(MOR) (CTAP, 0.09 mg/kg), k (KOR) (GNTI, 0.08 mg/kg) and δ (DOR) (NTI, 4 mg/kg) opioid receptors or antisense oligodeoxynucleotides directed against specific exons of the DOR, KOR and MOR (10 μg each probe/10 μl saline). i.c.v. administered were performed and the AWR score detected. The H2S (50 μM) In Vitro effects of H2S on MOR phosphorylation, β-arrestins recruitment and MOR internalization were determined in the neuronal cell line SKNMC and compared with those obtained with the MOR agonist DAMGO (1 μ). The effects of H2S and DAMGO on PI3K/AKT pathway was also detected. Results. H2S significantly inhibited the CRD-induced pain and spinal cFos overexpression. The H2S-induced analgesia was significantly reverted by naloxone, CTAP and antisense oligodeoxynucleotides against MOR, while both DOR and KOR inhibition appeared less effective. In the SKNMC cells, both H2S and DAMGO caused the timedependent MOR phosphorylation on serine 377 and the co-immunoprecipitation between MOR and β-arrestin, indicating that H2S induced the interaction of b-arrestins with MOR. Finally, plasma membrane fraction and immunoflurescence staining demonstrated that both DAMGO and H2S caused the translocation of MOR from the plasma membrane to the cytoplasma and that MOR internalization was inhibited by pre-treating cells with CTAP (1 μM). Both DAMGO and H2S induced AKT phosphorylation. CTAP inhibited the DAMGOinduced AKT phosphorylation but not those induced by H2S. Finally, pre-treating neuronal cells with the PI3K inhibitor LY294002 caused the blockade of H2S-induced MOR internalization. Conclusions. This study provides evidence that: 1) 2Sinhibits CRD-induced pain by interacting with peripheral and central MOR; 2) H2S causes MOR phosphorylation, the association between MOR and β-arrestins and MOR internalization; 3) H2S induces AKT phosphorylation; 4) inhibition of PI3K/AKT pathway results in the blockade of H2S-induced MOR internalization.

Morphine Reduces the Threshold of Helium Preconditioning Against Myocardial Infarction: The Role of Opioid Receptors in Rabbits

Brief, repetitive administration of helium before prolonged coronary artery occlusion and reperfusion protects myocardium against infarction. Opioid receptors mediate the cardioprotective effects of ischemic pre- and postconditioning, but whether these receptors also play a role in helium preconditioning is unknown. The authors tested the hypotheses that opioid receptors mediate helium preconditioning and that morphine (a mu(1)-opioid receptor agonist with delta(1)-opioid agonist properties) lowers the threshold of cardioprotection produced by helium in vivo. A randomized, prospective study. A university research laboratory. Male New Zealand white rabbits. Rabbits (n = 56) were instrumented for the measurement of systemic hemodynamics and subjected to a 30-minute left anterior descending coronary artery (LAD) occlusion and 3 hours of reperfusion. In separate experimental groups, rabbits (n = 6 or 7 per group) received 0.9% saline (control), 1 or 3 cycles of 70% helium-30% oxygen administered for 5 minutes interspersed with 5 minutes of an air-oxygen mixture, morphine (0.1 mg/kg intravenously), or the nonselective opioid antagonist naloxone (6 mg/kg intravenously) before LAD occlusion. Other groups of rabbits received 3 cycles of helium or 1 cycle of helium plus morphine (0.1 mg/kg) in the absence or presence of naloxone (6 mg/kg) before ischemia and reperfusion. Statistical analysis of data was performed with analysis of variance for repeated measures followed by Bonferroni modification of the Student t test. Myocardial infarct size was determined by using triphenyltetrazolium chloride staining and presented as a percentage of the left ventricular area at risk. Helium reduced myocardial infarct size in an exposure-related manner (36 +/- 6 [p > 0.05] and 25% +/- 4% [p < 0.05 v control] for 1 and 3 cycles of helium, respectively; data are mean +/- standard deviation) compared with control (44% +/- 7%). Morphine and naloxone alone did not affect infarct size (45 +/- 2 and 40% +/- 8%, respectively). The combination of 1 cycle of helium and morphine reduced infarct size (24% +/- 5%, p < 0.05 v control) to an equivalent degree as 3 cycles of helium. Naloxone pretreatment abolished cardioprotection produced by 3 cycles of helium (47% +/- 2%) and the combination of 1 cycle of helium plus morphine (45% +/- 4%). The results indicate that morphine lowers the threshold of helium preconditioning. Opioid receptors mediate helium preconditioning and its augmentation by morphine in vivo.

Antinociceptive effects of intracerebroventricular administration of guanine-based purines in mice: Evidences for the mechanism of action

It is well known that adenine-based purines exert multiple effects on pain transmission. However, less attention has been given to the potential effects of guanine-based purines (GBPs) on pain transmission. The aim of this study was to investigate the effects of intracerebroventricular (i.c.v.) guanosine and GMP on mice pain models. Mice received an i.c.v. injection of vehicle (saline or 10 μM NaOH), guanosine (5 to 400 nmol), or GMP (240 to 960 nmol). Additional groups were also pre-treated with i.c.v. injection of the A 1/A 2A antagonist caffeine (15 nmol), the non-selective opioid antagonist naloxone (12.5 nmol), or the 5'-nucleotidase inhibitor AOPCP (1 nmol). Measurements of CSF purine levels and cortical glutamate uptake were performed after treatments. Guanosine and GMP produced dose-dependent antinociceptive effects. Neither caffeine nor naloxone affected guanosine antinociception. Pre-treatment with AOPCP completely prevented GMP antinociception, indicating that conversion of GMP to guanosine is required for its antinociceptive effects. Intracerebroventricular administration of guanosine and GMP induced, respectively, a 180- and 1800-fold increase on CSF guanosine levels. Guanosine was able to prevent the decrease on cortical glutamate uptake induced by intraplantar capsaicin. This study provides new evidence on the mechanism of action of GBPs, with guanosine and GMP presenting antinociceptive effects in mice. This effect seems to be independent of adenosine and opioid receptors; it is, however, at least partially associated with modulation of the glutamatergic system by guanosine.