enkephalin--ala(2)-mephe(4)-gly(5)- and Respiratory-Insufficiency

The role of the different components of the respiratory network in the mediation of opioid-induced respiratory depression is still unclear. We investigated the contribution of the preBötzinger Complex (preBötC) and the neighbouring Bötzinger Complex (BötC) and inspiratory portion of the ventral respiratory group (iVRG) in anesthetized, vagotomized, paralyzed and artificially ventilated adult rabbits making use of bilateral microinjections (30-50 nl) of the μ-opioid receptor agonist [D-Ala

Topics: Analgesics, Opioid; Animals; Apnea; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Medulla Oblongata; Microinjections; Naloxone; Narcotic Antagonists; Neurons; Phrenic Nerve; Rabbits; Receptors, Opioid, mu; Respiratory Center; Respiratory Insufficiency

Drugs acting on μ-opioid receptors (MORs) are widely used as analgesics but present side effects including life-threatening respiratory depression. MORs are G-protein-coupled receptors inhibiting neuronal activity through calcium channels, adenylyl cyclase, and/or G-protein-gated inwardly rectifying potassium (GIRK) channels. The pathways underlying MOR-dependent inhibition of rhythmic breathing are unknown.. By using a combination of genetic, pharmacological, and physiological tools in rodents in vivo, the authors aimed to identify the role of GIRK channels in MOR-mediated inhibition of respiratory circuits.. GIRK channels were expressed in the ventrolateral medulla, a neuronal population regulating rhythmic breathing, and GIRK channel activation with flupirtine reduced respiratory rate in rats (percentage of baseline rate in mean ± SD: 79.4 ± 7.4%, n = 7), wild-type mice (82.6 ± 3.8%, n = 3), but not in mice lacking the GIRK2 subunit, an integral subunit of neuronal GIRK channels (GIRK2, 101.0 ± 1.9%, n = 3). Application of the MOR agonist [D-Ala, N-MePhe, Gly-ol]-enkephalin (DAMGO) to the ventrolateral medulla depressed respiratory rate, an effect partially reversed by the GIRK channel blocker Tertiapin-Q (baseline: 42.1 ± 7.4 breath/min, DAMGO: 26.1 ± 13.4 breath/min, Tertiapin-Q + DAMGO: 33.9 ± 9.8 breath/min, n = 4). Importantly, DAMGO applied to the ventrolateral medulla failed to reduce rhythmic breathing in GIRK2 mice (percentage of baseline rate: 103.2 ± 12.1%, n = 4), whereas it considerably reduced rate in wild-type mice (62.5 ± 17.7% of baseline, n = 4). Respiratory rate depression by systemic injection of the opioid analgesic fentanyl was markedly reduced in GIRK2 (percentage of baseline: 12.8 ± 15.8%, n = 5) compared with wild-type mice (72.9 ± 27.3%).. Overall, these results identify that GIRK channels contribute to respiratory inhibition by MOR, an essential step toward understanding respiratory depression by opioids.

Topics: Analgesics, Opioid; Animals; Bee Venoms; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Female; G Protein-Coupled Inwardly-Rectifying Potassium Channels; Male; Mice; Mice, Knockout; Rats; Rats, Wistar; Receptors, Opioid, mu; Respiratory Insufficiency

The preBötzinger Complex (preBC) plays an important role in respiratory rhythm generation. This study was designed to determine whether the preBC mediated opioid-induced respiratory rate depression at clinically relevant opioid concentrations in vivo and whether this role was age dependent.. Studies were performed in 22 young and 32 adult New Zealand White rabbits. Animals were anesthetized, mechanically ventilated, and decerebrated. The preBC was identified by the tachypneic response to injection of D,L-homocysteic acid. (1) The μ-opioid receptor agonist [D-Ala2,N-Me-Phe4,Gly-ol]-enkephalin (DAMGO, 100 μM) was microinjected into the bilateral preBC and reversed with naloxone (1 mM) injection into the preBC. (2) Respiratory depression was achieved with intravenous remifentanil (0.08 to 0.5 μg kg(-1) min(-1)). Naloxone (1 mM) was microinjected into the preBC in an attempt to reverse the respiratory depression.. (1) DAMGO injection depressed respiratory rate by 6 ± 8 breaths/min in young and adult rabbits (mean ± SD, P < 0.001). DAMGO shortened the inspiratory and lengthened the expiratory fraction of the respiratory cycle by 0.24 ± 0.2 in adult and young animals (P < 0.001). (2) During intravenous remifentanil infusion, local injection of naloxone into the preBC partially reversed the decrease in inspiratory fraction/increase in expiratory fraction in young and adult animals (0.14 ± 0.14, P < 0.001), but not the depression of respiratory rate (P = 0.19). PreBC injections did not affect respiratory drive. In adult rabbits, the contribution of non-preBC inputs to expiratory phase duration was larger than preBC inputs (3.5 [-5.2 to 1.1], median [25 to 75%], P = 0.04).. Systemic opioid effects on respiratory phase timing can be partially reversed in the preBC without reversing the depression of respiratory rate.

Topics: Aging; Analgesics, Opioid; Animals; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Female; Homocysteine; Male; Naloxone; Narcotic Antagonists; Neurons; Rabbits; Respiratory Insufficiency

Topics: Analgesics, Opioid; Animals; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Humans; Medulla Oblongata; Models, Neurological; Motor Neurons; Receptors, Opioid, mu; Respiratory Center; Respiratory Insufficiency; Respiratory Rate

Topics: Analgesics, Opioid; Animals; Brain Stem; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Humans; Hypercapnia; Hypoxia; Medulla Oblongata; Models, Neurological; Motor Neurons; Pons; Receptors, Opioid, mu; Respiratory Center; Respiratory Insufficiency; Respiratory Rate

Opioids activate glia in the central nervous system in part by activating the toll-like receptor 4 (TLR4)/myeloid differentiation 2 (MD2) complex. TLR4/MD2-mediated activation of glia by opioids compromises their analgesic actions. Glial activation is also hypothesized as pivotal in opioid-mediated reward and tolerance and as a contributor to opioid-mediated respiratory depression. We tested the contribution of TLR4 to opioid-induced respiratory depression using rhythmically active medullary slices that contain the pre-Bötzinger Complex (preBötC, an important site of respiratory rhythm generation) and adult rats in vivo. Injection with DAMGO (μ-opioid receptor agonist; 50 μM) or bath application of DAMGO (500 nM) or fentanyl (1 μM) slowed frequency recorded from XII nerves to 40%, 40%, or 50% of control, respectively. This DAMGO-mediated frequency inhibition was unaffected by preapplication of lipopolysaccharides from Rhodobacter sphaeroides (a TLR4 antagonist, 2,000 ng/ml) or (+)naloxone (1-10 μM, a TLR4-antagonist). Bath application of (-)naloxone (500 nM; a TLR4 and μ-opioid antagonist), however, rapidly reversed the opioid-mediated frequency decrease. We also compared the opioid-induced respiratory depression in slices in vitro in the absence and presence of bath-applied minocycline (an inhibitor of microglial activation) and in slices prepared from mice injected (ip) 18 h earlier with minocycline or saline. Minocycline had no effect on respiratory depression in vitro. Finally, the respiratory depression evoked in anesthetized rats by tail vein infusion of fentanyl was unaffected by subsequent injection of (+)naloxone, but completely reversed by (-)naloxone. These data indicate that neither activation of microglia in preBötC nor TLR4/MD2-activation contribute to opioid-induced respiratory depression.

Topics: Analgesics, Opioid; Animals; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Female; Fentanyl; Male; Medulla Oblongata; Minocycline; Naloxone; Neuroglia; Rats; Rats, Sprague-Dawley; Receptors, Opioid, mu; Respiration; Respiratory Insufficiency; Signal Transduction; Toll-Like Receptor 4

Aminomorphinans are a relatively young class of opioid drugs among which substances of high in vitro efficacy and favorable in vivo action are found. We report the synthesis and pharmacological evaluation of novel 6β-acylaminomorphinans. 6β-Morphinamine and 6β-codeinamine were stereoselectively synthesized by Mitsunobu reaction. The aminomorphinans were subsequently acylated with diversely substituted cinnamic acids. In vitro binding studies on cinnamoyl morphinamines showed moderate affinity for all opiate receptors with some selectivity for mu opioid receptors, while cinnamoyl codeinamines only showed affinity for mu opioid receptors. In vivo analgesia studies showed significant analgesic activity of 6β-cinnamoylmorphinamine mediated by mu and delta receptors. The lead compound was found to be roughly equipotent to morphine (ED₅₀ 3.13 ± 1.09 mg/kg) but devoid of the dangerous side-effect respiratory depression, a major issue associated with traditional opioid therapy.

Topics: Analgesics; Animals; Dose-Response Relationship, Drug; Hot Temperature; Mice; Molecular Structure; Morphinans; Morphine; Narcotic Antagonists; Pain Measurement; Pain Threshold; Receptors, Opioid; Respiratory Insufficiency; Structure-Activity Relationship; Time Factors

Respiratory depression is a therapy-limiting side effect of opioid analgesics, yet our understanding of the brain circuits mediating this potentially lethal outcome remains incomplete. Here we studied the contribution of the rostral ventromedial medulla (RVM), a region long implicated in pain modulation and homeostatic regulation, to opioid-induced respiratory depression. Microinjection of the μ-opioid agonist DAMGO in the RVM of lightly anesthetized rats produced both analgesia and respiratory depression, showing that neurons in this region can modulate breathing. Blocking opioid action in the RVM by microinjecting the opioid antagonist naltrexone reversed the analgesic and respiratory effects of systemically administered morphine, showing that this region plays a role in both the analgesic and respiratory-depressant properties of systemically administered morphine. The distribution of neurons directly inhibited by RVM opioid microinjection was determined with a fluorescent opioid peptide, dermorphin-Alexa 594, and found to be concentrated in and around the RVM. The non-opioid analgesic improgan, like DAMGO, produced antinociception but, unlike DAMGO, stimulated breathing when microinjected into the RVM. Concurrent recording of RVM neurons during improgan microinjection showed that this agent activated RVM ON-cells, OFF-cells, and NEUTRAL-cells. Since opioids are known to activate OFF-cells but suppress ON-cell firing, the differential respiratory response to these two analgesic drugs is best explained by their opposing effects on the activity of RVM ON-cells. These findings show that pain relief can be separated pharmacologically from respiratory depression and identify RVM OFF-cells as important central targets for continued development of potent analgesics with fewer side effects.

Topics: Analgesics, Opioid; Animals; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Evoked Potentials; Male; Medulla Oblongata; Morphine; Naltrexone; Narcotic Antagonists; Neurons; Nociception; Nociceptive Pain; Rats; Rats, Sprague-Dawley; Respiratory Insufficiency

Hypothetic mechanisms for respirogenic methylxanthine actions include blockade of adenosine receptors or phosphodiesterase-4 (PDE4) in inspiratory pre-Bötzinger complex (preBötC) networks. Here, we studied this by analyzing stimulating caffeine and theophylline actions on mu-opioid-depressed inspiratory-related rhythm in the ventrolateral aspect of rat brainstem slices. The methylxanthines restored DAMGO (0.5-1 microM) depressed rhythm only at >1mM, which is approximately 10-fold higher than selective for adenosine receptors. Adenosine receptor blockers did neither counter DAMGO inhibition nor change control rhythm, similar to adenosine (0.1-2.5 mM). The specific PDE4 blocker rolipram (5 microM) restored DAMGO-depressed rhythm incompletely, but effectively reversed similar inhibition by clinical mu-agonist (fentanyl, 0.1 microM). At 0.25 microM, rolipram boosted incomplete recovery by 1 mM theophylline of DAMGO-depressed rhythm. Findings indicate that methylxanthines excite rhythmogenic preBötC networks via blockade of cAMP dependent PDE4 and suggest that specific PDE4 inhibitors (plus low methylxanthine doses) stimulate breathing effectively. We discuss why methylxanthine doses for preBötC stimulation need to be higher than those for respirogenic effects in vivo.

Topics: Adenosine A1 Receptor Antagonists; Adenosine A2 Receptor Antagonists; Analgesics, Opioid; Animals; Animals, Newborn; Caffeine; Electrophysiology; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Fentanyl; In Vitro Techniques; Interneurons; Nerve Net; Phosphodiesterase 4 Inhibitors; Phosphodiesterase Inhibitors; Purinergic P1 Receptor Antagonists; Rats; Rats, Sprague-Dawley; Rats, Wistar; Receptors, Opioid, mu; Respiratory Insufficiency; Respiratory Physiological Phenomena; Rolipram; Theophylline; Xanthines

The raphe magnus (RM) participates in opioid analgesia and contains pain-modulatory neurons with respiration-related discharge. Here, we asked whether RM contributes to respiratory depression, the most prevalent lethal effect of opioids. To investigate whether opioidergic transmission in RM produces respiratory depression, we microinjected a mu-opioid receptor agonist, DAMGO, or morphine into the RM of awake rodents. In mice, opioid microinjection produced sustained decreases in respiratory rate (170 to 120 breaths/min), as well as heart rate (520 to 400 beats/min). Respiratory sinus arrhythmia, indicative of enhanced parasympathetic activity, was prevalent in mice receiving DAMGO microinjection. We performed similar experiments in rats but observed no changes in breathing rate or heart rate. Both rats and mice experienced significantly more episodes of bradypnea, indicative of impaired respiratory drive, after opioid microinjection. During spontaneous arousals, rats showed less tachycardia after opioid microinjection than before microinjection, suggestive of an attenuated sympathetic tone. Thus, activation of opioidergic signaling within RM produces effects beyond analgesia, including the unwanted destabilization of cardiorespiratory function. These adverse effects on homeostasis consequent to opioid microinjection imply a role for RM in regulating the balance of sympathetic and parasympathetic tone.

Topics: Analgesics, Opioid; Animals; Arrhythmia, Sinus; Bradycardia; Cardiovascular System; Disease Models, Animal; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Heart Rate; Homeostasis; Male; Mice; Mice, Inbred C57BL; Microinjections; Morphine; Motor Activity; Raphe Nuclei; Rats; Rats, Sprague-Dawley; Respiratory Insufficiency; Respiratory System; Sleep

Opioids are among the most effective analgesics, but a major limitation for their therapeutic usefulness is their induction of respiratory depression. Endomorphin-1 (EM1), in contrast to several other mu opioids, exhibits a threshold for respiratory depression that is well above its threshold for analgesia. Its effect on sensitivity to CO(2), however, remains unknown. Minute ventilation (V(E)) in 2, 4, and 6% CO(2) was measured before and after systemic administration of EM1, endomorphin-2 (EM2), DAMGO, and morphine in the conscious rat. EM1 and EM2 attenuated the hypercapnic ventilatory response (HCVR) only in high doses, while DAMGO and morphine diminished the HCVR in much lower doses. The ventilatory effects of high doses of all 4 agonists were blocked by the mu-opioid antagonist naloxone (0.4 mg/kg i.v.), but not by the peripherally restricted mu-opioid antagonist, methyl-naloxone (0.4 mg/kg i.v.). It was concluded that the endomorphins attenuated the HCVR only in large doses, well beyond the analgesic threshold, and did so through a centrally mediated mu-opioid mechanism.

Topics: Adaptation, Physiological; Analgesics, Opioid; Animals; Carbon Dioxide; Dose-Response Relationship, Drug; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Hypercapnia; Male; Morphine; Naloxone; Oligopeptides; Pulmonary Ventilation; Rats; Rats, Sprague-Dawley; Receptors, Opioid, mu; Respiration; Respiratory Insufficiency

In adult pentobarbital-anesthetized and unanesthetized decerebrate cats, the D(1)R agonists (6-chloro-APB, SKF-38393, dihydrexidine) given intravenously restored phrenic nerve and vagus nerve respiratory discharges and firing of bulbar post-inspiratory neurons after the discharges were abolished by the micro-opioid receptor agonist fentanyl given intravenously. Reversal of opioid-mediated discharge depression was prevented by the D(1)R antagonist SCH23390. Iontophoresis of the micro-opioid receptor agonist DAMGO depressed firing of medullary bulbospinal inspiratory neurons. Co-iontophoresis of SKF-38393 did not restore firing and had no effect on bulbospinal inspiratory neuron discharges when applied alone. The D(1)R agonists given intravenously prolonged and intensified phrenic nerve and bulbospinal inspiratory neuron discharges. They also increased reactivity to CO(2) by lowering the phrenic nerve apnea threshold and shifting the phrenic nerve-CO(2) response curve to lower et(CO(2)) levels. Intravenous fentanyl on the other hand decreased CO(2) reactivity by shifting the phrenic nerve apnea threshold and the response curve to higher et(CO(2)) levels. Fentanyl effects on reactivity were partially reversed by D(1)R agonists.

Topics: Action Potentials; Analgesics, Opioid; Analysis of Variance; Animals; Apnea; Carbon Dioxide; Cats; Decerebrate State; Dopamine Agonists; Dose-Response Relationship, Drug; Drug Administration Routes; Drug Interactions; Electric Stimulation; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Fentanyl; Iontophoresis; Male; Medulla Oblongata; Neurons; Phrenic Nerve; Reaction Time; Receptors, Dopamine D1; Respiratory Insufficiency; Sensory Thresholds; Vagus Nerve

Mu-opioid receptor agonists depress tidal volume, decrease chest wall compliance, and increase upper airway resistance. In this study, potential neuronal sites and mechanisms responsible for the disturbances were investigated, dose-response relationships were established, and it was determined whether general anesthesia plays a role. Effects of micro-opioid agonists on membrane properties and discharges of respiratory bulbospinal, vagal, and propriobulbar neurons and phrenic nerve activity were measured in pentobarbital-anesthetized and unanesthetized decerebrate cats. In all types of respiratory neurons tested, threshold intravenous doses of the micro-opioid agonist fentanyl slowed discharge frequency and prolonged duration without altering peak discharge intensity. Larger doses postsynaptically depressed discharges of inspiratory bulbospinal and inspiratory propriobulbar neurons that might account for depression of tidal volume. Iontophoresis of the micro-opioid agonist DAMGO also depressed the intensity of inspiratory bulbospinal neuron discharges. Fentanyl given intravenously prolonged discharges leading to tonic firing of bulbospinal expiratory neurons in association with reduced hyperpolarizing synaptic drive potentials, perhaps explaining decreased inspiratory phase chest wall compliance. Lowest effective doses of fentanyl had similar effects on vagal postinspiratory (laryngeal adductor) motoneurons, whereas in vagal laryngeal abductor and pharyngeal constrictor motoneurons, depression of depolarizing synaptic drive potentials led to sparse, very-low-frequency discharges. Such effects on three types of vagal motoneurons might explain tonic vocal fold closure and pharyngeal obstruction of airflow. Measurements of membrane potential and input resistance suggest the effects on bulbospinal Aug-E neurons and vagal motoneurons are mediated presynaptically. Opioid effects on the respiratory neurons were similar in anesthetized and decerebrate preparations.

Topics: Action Potentials; Analgesics, Opioid; Animals; Cats; Decerebrate State; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Fentanyl; Inhalation; Iontophoresis; Medulla Oblongata; Neurons; Phrenic Nerve; Proprioception; Receptors, Opioid, mu; Respiratory Center; Respiratory Insufficiency; Spinal Cord; Vagus Nerve

The major side effect of morphine and its active metabolite, morphine-6-glucuronide (M6G), is respiratory depression, which is mediated by mu opioid receptors in the medulla and pons. Although the effect of morphine on coupling between mu opioid receptors and G proteins has been studied, the effect of M6G on this coupling has not. Therefore, stimulation of guanylyl-5'-O-([gamma(35)S]-thio)-triphosphate ([(35)S]-GTPgammaS) binding by these two narcotic analgesic drugs was compared to the mu-specific synthetic opioid peptide [D-Ala(2), N-MePhe(4), Gly-ol(5)]enkephalin in Chinese hamster ovarian cells stably transfected with the murine mu opioid receptor and in brainstem membranes prepared from 3-, 7-, and 14-day-old guinea pigs. All three agonists stimulated [(35)S]-GTPgammaS binding in transfected cells and neural tissue, and the stimulation was antagonized by naloxone. In brainstem membranes, but not transfected cells, M6G was less efficacious but more potent than morphine, which may be due to differences between murine and guinea pig mu opioid receptors or in the G proteins in these two tissues. Efficacy of the agonists did not change during development, but overall potency decreased between 3 and 14 days after birth. In vivo potency differences for respiratory depression between morphine and M6G are qualitatively similar to in vitro potency differences of these drugs to stimulate [(35)S]-GTPgammaS binding in neonatal guinea pig brainstem membranes. Tolerance to opioid effects on [(35)S]-GTPgammaS binding developed in transfected cells incubated with morphine with the maximum decrease in potency occurring 18 h later than the maximum decline in efficacy.

Topics: Analgesics, Opioid; Animals; Animals, Newborn; Binding Sites; Brain Stem; Cell Membrane; CHO Cells; Cricetinae; Drug Tolerance; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Guanosine 5'-O-(3-Thiotriphosphate); Guinea Pigs; Morphine; Morphine Derivatives; Naloxone; Narcotic Antagonists; Radioligand Assay; Receptors, Opioid, mu; Respiratory Insufficiency; Respiratory Physiological Phenomena; Subcellular Fractions; Sulfur Radioisotopes