Opioid action on respiratory neuron activity of the isolated respiratory network in newborn rats

BACKGROUND: Underlying mechanisms behind opioid-induced respiratory depression are not fully understood. The authors investigated changes in burst rate, intraburst firing frequency, membrane properties, as well as presynaptic and postsynaptic events of respiratory neurons in the isolated brainstem after administration of opioid receptor agonists. METHODS: Newborn rat brainstem-spinal cord preparations were used and superfused with mu-, kappa-, and delta-opioid receptor agonists. Whole cell recordings were performed from three major classes of respiratory neurons (inspiratory, preinspiratory, and expiratory). RESULTS: Mu- and kappa-opioid receptor agonists reduced the spontaneous burst activity of inspiratory neurons and the C4 nerve activity. Forty-two percent of the inspiratory neurons were hyperpolarized and decreased in membrane resistance during opioid-induced respiratory depression. Furthermore, under synaptic block by tetrodotoxin perfusion, similar changes of inspiratory neuronal membrane properties occurred after application of mu- and kappa-opioid receptor agonists. In contrast, resting membrane potential and membrane resistance of preinspiratory and majority of expiratory neurons were unchanged by opioid receptor agonists, even during tetrodotoxin perfusion. Simultaneous recordings of inspiratory and preinspiratory neuronal activities confirmed the selective inhibition of inspiratory neurons caused by mu- and kappa-opioid receptor agonists. Application of opioids reduced the slope of rising of excitatory postsynaptic potentials evoked by contralateral medulla stimulation, resulting in a prolongation of the latency of successive first action potential responses. CONCLUSIONS: Mu- and kappa-opioid receptor agonists caused reduction of final motor outputs by mainly inhibiting medullary inspiratory neuron network. This inhibition of inspiratory neurons seems to be a result of both a presynaptic and postsynaptic inhibition. The central respiratory rhythm as reflected by the preinspiratory neuron burst rate was essentially unaltered by the agonists.     

Differential Presynaptic Effects of Opioid Agonists on Aδ- and C-afferent Glutamatergic Transmission to the Spinal Dorsal Horn

Background: Although intrathecal administration of opioids produces antinociceptive effects in the spinal cord, it has not been established whether intrathecal opioid application more effectively terminates C fiber-mediated pain than A fiber-mediated pain. Here, the authors focus on the differences in opioid actions on A[delta]- and C-afferent responses.

Methods: Using the whole cell patch clamp technique, the authors investigated the presynaptic inhibitory actions of [mu]-, [delta]-, and [kappa]-opioid receptor agonists on primary afferent-evoked excitatory postsynaptic currents (EPSCs) in substantia gelatinosa neurons of adult rat spinal cord slices.

Results: The [mu] agonist DAMGO (0.1, 1 [mu]m) reduced the amplitude of glutamatergic monosynaptic A[delta]- or C fiber-evoked EPSCs. C fiber-evoked EPSCs were inhibited to a greater extent than A[delta] fiber-evoked EPSCs. The [delta] agonist DPDPE (1, 10 [mu]m) produced modest inhibition of A[delta]- or C fiber-evoked EPSCs. In contrast, the [kappa] agonist U69593 (1 [mu]m) did not affect the amplitude of either A[delta] or C fiber-evoked EPSCs.     

Nerve Injury Induces a Tonic Bilateral μ-Opioid Receptor-mediated Inhibitory Effect on Mechanical Allodynia in Mice

Background: Mice lacking the [mu]-opioid receptor gene have been used to characterize the role of [mu]-opioid receptors in nociception and the analgesic actions of opioid agonists. In this study, the authors determined the role of [mu]-opioid receptors in neuropathic pain behaviors and the effectiveness of [mu]- and [kappa]-opioid receptor agonists on this behavior in mice.

Methods: The authors studied the behavioral responses of [mu]-opioid receptor knockout and wild-type mice to thermal and mechanical stimuli before and after neuropathic pain induced by unilateral ligation and section of the L5 spinal nerve. Response to mechanical stimuli was evaluated by determining the frequency of hind paw withdrawal to repetitive stimulation using a series of von Frey monofilaments. Thermal hyperalgesia was assessed by determining the paw withdrawal latencies to radiant heat and frequency of hind paw withdrawal to cooling stimuli. The effects of systemic morphine, the [kappa]-opioid agonist U50488H, and naloxone on responses to mechanical and thermal stimuli were also studied in spinal nerve-injured mice.

Results: After spinal nerve injury, wild-type mice developed increased responsiveness to mechanical, heat, and cooling stimuli ipsilateral to nerve injury. [mu]-Opioid receptor knockout mice not only had more prominent mechanical allodynia in the nerve-injured paw, but also expressed contralateral allodynia to mechanical stimuli. Hyperalgesia to thermal stimuli was similar between [mu]-opioid knockout and wild-type animals. Morphine decreased mechanical allodynia dose dependently (3-30 mg/kg subcutaneous) in wild-type mice-an effect that was attenuated in the heterozygous mice and absent in the homozygous [mu]-opioid knockout mice. The [kappa]-opioid agonist U50488H (3-10 mg/kg subcutaneous) attenuated mechanical allodynia in wild-type, heterozygous, and homozygous [mu]-opioid mice. Naloxone in wild-type mice resulted in enhanced ipsilateral and contralateral allodynia to mechanical stimuli that resembled the pain behavior observed in [mu]-opioid receptor knockout mice.     

Enhancement of Spinal N-Methyl-d-aspartate Receptor Function by Remifentanil Action at [delta]-Opioid Receptors as a Mechanism for Acute Opioid-induced Hyperalgesia or Tolerance

Background: Intraoperative remifentanil infusions have been associated with postoperative opioid-induced hyperalgesia and tolerance. Using a previously identified subpopulation of spinal neurons that displays an augmentation in N-methyl-d-aspartate (NMDA) receptor current after chronic morphine, investigations were undertaken to determine whether remifentanil induces acute increases in NMDA responses that are concentration dependent and receptor subtype dependent.

Methods: Electrophysiologic recordings of NMDA current were made from cultured rat dorsal horn neurons treated with remifentanil at various concentrations for 60 min. Selective [mu]- or [delta]-opioid receptor inhibitors and agonists were used to determine the site of action of remifentanil.

Results: Remifentanil at 4, 6, and 8 nm, but not higher or lower concentrations, caused significant mean increases in NMDA peak current amplitude of 37.30% (P < 0.001), 30.19% (P < 0.001), and 23.52% (P = 0.025), respectively, over control conditions. This occurred by 36 min of remifentanil perfusion and persisted throughout its washout. Inhibition by 100 nm naloxone or 1 nm naltrindole attenuated the remifentanil-induced NMDA response increase. Selective [delta]-opioid agonists [D-Pen2, D-Pen5]enkephalin and deltorphin II displayed a similar bell-shaped concentration-response relation for the enhancement of NMDA responses, and 10 nm deltorphin II occluded the effects of 4 nm remifentanil on NMDA responses.     

A neuronal mechanism of propofol-induced central respiratory depression in newborn rats

The neural mechanisms of propofol-induced central respiratory depression remain poorly understood. In the present study, we studied these mechanisms and the involvement of gamma-aminobutyric acid (GABA)A receptors in propofol-induced central respiratory depression. The brainstem and the cervical spinal cord of 1- to 4-day-old rats were isolated, and preparations were maintained in vitro with oxygenated artificial cerebrospinal fluid. Rhythmic inspiratory burst activity was recorded from the C4 spinal ventral root. The activity of respiratory neurons in the ventrolateral medulla was recorded using a perforated patch-clamp technique. We found that bath-applied propofol decreased C4 inspiratory burst rate, which could be reversed by the administration of a GABAA antagonist, bicuculline. Propofol caused resting membrane potentials to hyperpolarize and suppressed the firing of action potentials in preinspiratory and expiratory neurons. In contrast, propofol had little effect on resting membrane potentials and action potential firing in inspiratory neurons. Our findings suggest that the depressive effects of propofol are, at least in part, mediated by the agonistic action of propofol on GABAA receptors. It is likely that the GABAA receptor-mediated hyperpolarization of preinspiratory neurons serves as the neuronal basis of propofol-induced respiratory depression in the newborn rat.     

Activation of μ- and δ-Opioid Receptors Causes Presynaptic Inhibition of Glutamatergic Excitation in Neocortical Neurons

Background: The mechanism underlying the depressant effect of opioids on neuronal activity within the neocortex is still not clear. Three modes of action have been suggested: (1) inhibition by activation of postsynaptic potassium channels, (2) interaction with postsynaptic glutamate receptors, and (3) presynaptic inhibition of glutamate release. To address this issue, the authors investigated the effects of [mu]- and [delta]-receptor agonists on excitatory postsynaptic currents (EPSCs) and on membrane properties of neocortical neurons.

Methods: Intracellular recordings were performed in rat brain slices. Stimulus-evoked EPSCs mediated by different glutamate receptor subtypes were pharmacologically isolated, and opioids were applied by addition to the bathing medium. Possible postsynaptic interactions between glutamate and opioid receptors were investigated using microiontophoretic application of glutamate on neurons functionally isolated from presynaptic input.

Results: [delta]-Receptor activation by d-Ala2-d-Leu5-enkephalin (DADLE) reduced the amplitudes of EPSCs by maximum 60% in a naltrindole-reversible manner (EC50: 6-15 nm). In 30-40% of the neurons investigated, higher concentrations (0.1-1 [mu]m) of DADLE activated small outward currents. The [mu]-receptor selective agonist d-Ala2-N-MePhe5-Gly5-ol-enkephalin (0.1-1 [mu]m) depressed the amplitudes of EPSCs by maximum 30% without changes in postsynaptic membrane properties. In the absence of synaptic transmission, inward currents induced by microiontophoretic application of glutamate were not affected by DADLE.     

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

Background: 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-Ala2, N-Me-Phe4, Gly5-ol]-enkephalin (DAMGO) on SG neurons with different intrinsic firing properties.

Methods: Whole cell patch clamp recordings from spinal cord slices of Wistar rats were used to study the sensitivity of SG neurons to DAMGO.

Results: Three groups of neurons with distinct distributions in SG were classified: tonic-, adapting-, and delayed-firing neurons. DAMGO at 1 [mu]m 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 [mu]m Ba2+ and 500 [mu]m Cs+ but increased by 50 [mu]m baclofen. As a functional consequence of DAMGO action, a majority of tonic-firing neurons changed their pattern of intrinsic firing from tonic to adapting.     

Subpopulation of Dorsal Horn Neurons Displays Enhanced N-methyl-d-aspartate Receptor Function after Chronic Morphine Exposure

Background: Morphine tolerance may be attributed to enhancement of glutamatergic neurotransmission, in particular to increased function of the N-methyl-d-aspartate (NMDA) receptor. The cellular mechanisms responsible for these changes remain poorly defined. The authors identified and characterized a specific subpopulation of dorsal horn neurons, displaying NMDA receptor plasticity in response to chronic morphine administration.

Methods: The authors undertook current clamped and voltage clamped recordings of NMDA receptor-mediated responses from cultured rat dorsal horn neurons that were untreated or treated for 7 days with 1 or 100 [mu]m morphine.

Results: Smaller (capacitance <= 22 pF), tonic firing neurons showed a significantly enhanced NMDA receptor-mediated peak current after prolonged morphine treatment, whereas larger and phasic firing neurons showed no enhancement. With high-concentration but not low-concentration morphine treatment, Mg2+ blockade of NMDA receptors at resting membrane potentials was reduced. Furthermore, the chronic opioid-induced increase in NMDA current was attenuated by pretreatment with either a [mu]-opioid receptor inhibitor (naloxone) or an NMDA receptor inhibitor (2-amino-5-phosphonovalerate) (low-concentration > high-concentration morphine).     

In Vitro Electrophysiologic Effects of Morphine in Rabbit Ventricular Myocytes

Background: Morphine is widely used in patients undergoing surgical operations and is also reported to mediate cardioprotection of preconditioning. The current study determined effects of morphine at therapeutic to pharmacologic concentrations on cardiac action potential, L-type Ca2+ current (ICa.L), delayed rectifier K+ current (IK), and inward rectifier K+ current (IK1) in isolated rabbit ventricular myocytes.

Methods: Ventricular myocytes were enzymatically isolated from rabbit hearts. Action potential and membrane currents were recorded in current and voltage clamp modes.

Results: Morphine at concentrations from 0.01 to 1 [mu]m significantly prolonged cardiac action potential, and at 0.1 and 1 [mu]m slightly but significantly hyperpolarized the resting membrane potential. In addition, morphine at 0.1 [mu]m significantly augmented ICa.L (at +10 mV) from 5.9 +/- 1.9 to 7.3 +/- 1.7 pA/pF (by 23%; P < 0.05 vs. control) and increased IK1 (at -60 mV) from 2.8 +/- 1.0 to 3.5 +/- 0.9 pA/pF (by 27%; P < 0.05 vs. control). Five [mu]m naltrindole (a selective [delta]-opioid receptor antagonist) or 5 [mu]m norbinaltorphimine (a selective [kappa]-opioid receptor antagonist) prevented the increase in ICa.L induced by morphine, but 5 [mu]m CTOP (a selective [mu]-opioid receptor antagonist) did not. The three types of opioid antagonists did not affect the augmentation of IK1 by morphine. Morphine had no effect on IK.     

Morphine Preconditions Purkinje Cells against Cell Death under In Vitro Simulated Ischemia-Reperfusion Conditions

Background: Morphine pretreatment via activation of [delta]1-opioid receptors induces cardioprotection. In this study, the authors determined whether morphine preconditioning induces ischemic tolerance in neurons.

Methods: Cerebellar brain slices from adult Sprague-Dawley rats were incubated with morphine at 0.1-10 [mu]m in the presence or absence of various antagonists for 30 min. They were then kept in morphine- and antagonist-free buffer for 30 min before they were subjected to simulated ischemia (oxygen-glucose deprivation) for 20 min. After being recovered in oxygenated artificial cerebrospinal fluid for 5 h, they were fixed for morphologic examination to determine the percentage of undamaged Purkinje cells.

Results: The survival rate of Purkinje cells was significantly higher in slices preconditioned with morphine (>= 0.3 [mu]m) before the oxygen-glucose deprivation (57 +/- 4% at 0.3 [mu]m morphine) than that of the oxygen-glucose deprivation alone (39 +/- 3%, P < 0.05). This morphine preconditioning-induced neuroprotection was abolished by naloxone, a non-type-selective opioid receptor antagonist, by naltrindole, a selective [delta]-opioid receptor antagonist, or by 7-benzylidenenaltrexone, a selective [delta]1-opioid receptor antagonist. However, the effects were not blocked by the [mu]-, [kappa]-, or [delta]2-opioid receptor antagonists, [beta]-funaltrexamine, nor-binaltorphimine, or naltriben, respectively. Morphine preconditioning-induced neuroprotection was partially blocked by the selective mitochondrial adenosine triphosphate-sensitive potassium channel antagonist, 5-hydroxydecanoate, or the mitochondrial electron transport inhibitor, myxothiazol. None of the inhibitors used in this study alone affected the simulated ischemia-induced neuronal death.     

Norepinephrine Facilitates Inhibitory Transmission in Substantia Gelatinosa of Adult Rat Spinal Cord (Part 2): Effects on Somatodendritic Sites of GABAergic Neurons

Background: It has been reported previously that norepinephrine, when applied to the spinal cord dorsal horn, excites a subpopulation of dorsal horn neurons, presumably inhibitory interneurons. In the current study, the authors tested whether norepinephrine could activate inhibitory interneurons, specifically those that are "GABAergic."

Methods: A transverse slice was obtained from a segment of the lumbar spinal cord isolated from adult male Sprague-Dawley rats. Whole-cell patch-clamp recordings were made from substantia gelatinosa neurons using the blind patch-clamp technique. The effects of norepinephrine on spontaneous GABAergic inhibitory postsynaptic currents were studied.

Results: In the majority of substantia gelatinosa neurons tested, norepinephrine (10-60 [mu]M) significantly increased both the frequency and the amplitude of GABAergic inhibitory postsynaptic currents. These increases were blocked by tetrodotoxin (1 [mu]M). The effects of norepinephrine were mimicked by the [alpha]1-receptor agonist phenylephrine (10-80 [mu]M) and inhibited by the [alpha]1-receptor antagonist WB-4101 (0.5 [mu]M). Primary-afferent-evoked polysynaptic excitatory postsynaptic potentials or excitatory postsynaptic currents in wide-dynamic-range neurons of the deep dorsal horn were also attenuated by phenylephrine (40 [mu]M).     

Remifentanil Preconditioning Protects against Ischemic Injury in the Intact Rat Heart

Background: Opioid receptors mediate cardiac ischemic preconditioning. Remifentanil is a new, potent ultra-short-acting phenylpiperidine opioid used in high doses for anesthesia. The authors hypothesize that pretreatment with this drug confers cardioprotection.

Methods: Male Sprague-Dawley rats were anesthetized and the chest was opened. All animals were subjected to 30 min of occlusion of the left coronary artery and 2 h of reperfusion. Before the 30-min occlusion, rats received either preconditioning by ischemia (ischemic preconditioning, 5-min occlusion, 5-min reperfusion x 3) or pretreatment with remifentanil, performed with the same regime (3 x 5-min infusions) using 0.2, 0.6, 2, 6, or 20 [mu]g[middle dot]kg-1[middle dot]min-1 intravenously. The experiment was repeated with naltrindole (a selective [DELTA]-opioid receptor antagonist, 5 mg/kg), nor-binaltorphimine (a selective [kappa]-OR antagonist, 5 mg/kg), or CTOP (a selective [mu]-opioid receptor antagonist, 1 mg/kg) administered before remifentanil-induced preconditioning or ischemic preconditioning, respectively. Infarct size, as a percentage of the area at risk, was determined by 2,3,5-triphenyltetrazolium staining.

Results: There was a dose-related reduction in infarct size/area at risk after treatment with remifentanil that was similar to that seen with ischemic preconditioning. This effect was prevented or significantly attenuated by coadministration of a [mu], [kappa], or [DELTA]-opioid antagonist. The infarct-sparing effect of ischemic preconditioning was abolished by blockade of [kappa]-opioid receptors or [DELTA]-opioid receptors but not by [mu]-opioid receptors.     

Salicylate action on medullary inspiratory neuron activity in a brainstem-spinal cord preparation from newborn rats

Salicylate affects central respiratory control. The inspiratory neurons are the most important component of the medullary respiratory control center because they modulate the final motor output via the phrenic nerve. We investigated changes in burst rate, intraburst firing frequency, and membrane properties of inspiratory neurons in the isolated brainstem after the administration of salicylate. Newborn rat brainstem-spinal cord preparations were superfused with salicylate. Whole-cell recordings were performed from inspiratory neurons. Application of 1 mM salicylate caused an increase in the inspiratory neuronal burst rate from 6.9 +/- 1.6 bursts/min to 8.2 +/- 1.9 bursts/min (P < 0.05). The inspiratory neuron burst rate decreased from 8.3 +/- 0.7 bursts/min to 4.5 +/- 1.1 bursts/min after the application of 10 mM salicylate (P < 0.01). The depressant effect of 10 mM salicylate was antagonized by the gamma-aminobutyric acid (GABA) receptor antagonist bicuculline (1 microM). Resting membrane potential and intraburst firing frequency did not change with the application of salicylate and bicuculline even when the burst rate did change. We conclude that the effects of salicylate on the medullary inspiratory neurons are mainly due to a presynaptic action. GABAergic mechanisms are probably involved in the salicylate-induced central respiratory depression.     

Sympathetic Neural Activation Evoked by μ-Receptor Blockade in Patients Addicted to Opioids Is Abolished by Intravenous Clonidine

Background: [mu]-Opioid receptor blockade by naloxone administered for acute detoxification in patients addicted to opioids markedly increases catecholamine plasma concentrations, muscle sympathetic activity (MSA), and is associated with cardiovascular stimulation despite general anesthesia. The current authors tested the hypothesis that the [alpha]2-adrenoceptor agonist clonidine (1) attenuates increased MSA during [mu]-opioid receptor blockade for detoxification, and (2) prevents cardiovascular activation when given before detoxification.

Methods: Fourteen mono-opioid addicted patients received naloxone during propofol anesthesia. Clonidine (10 [mu]g kg-1 administered over 5 min + 5 [mu]g kg-1 h-1 intravenous) was infused either before (n = 6) or after (n = 6) naloxone administration. Two patients without immediate clonidine administration occurring after naloxone administration served as time controls. Muscle sympathetic activity (n = 8) in the peroneal nerve, catecholamine plasma concentrations (n = 14), arterial blood pressure, and heart rate were assessed in awake patients, during propofol anesthesia before and after [mu]-opioid receptor blockade, and after clonidine administration.

Results: [mu]-Receptor blockade markedly increased MSA from a low activity (burst frequency: from 2 burst/min +/- 1 to 24 +/- 8, means +/- SD). Similarly, norepinephrine (41 pg/ml +/- 37 to 321 +/- 134) and epinephrine plasma concentration (13 pg/ml +/- 6 to 627 +/- 146) significantly increased, and were associated with, increased arterial blood pressure and heart rate. Clonidine immediately abolished both increased MSA (P < 0.001) and catecholamine plasma concentrations (P < 0.001). When clonidine was given before [mu]-opioid receptor blockade, catecholamine plasma concentrations and hemodynamic variables did not change.     

Pharmacology of Opioid Inhibition to Noxious Uterine Cervical Distension

Background: Reflex abdominal muscle contraction elicited by colorectal distension in male rats is inhibited by [mu]- and [kappa]-opioid receptor agonists and sites of action and receptor subtypes have been probed. The authors examined the pharmacology of opioid agonist inhibition in visceral pain related to the uterine cervix, the source of labor pain.

Methods: Ovariectomized female rats were anesthetized with halothane, and metal rods inserted in the uterine cervix through a small midline laparotomy. After a period of stabilization the cervix was distended by manual separation of the rods, using stimuli of 25-100 g, and reflex rectus abdominis electromyographic activity was recorded. After determining the stimulus response relationship, we tested inhibition of reflex activity by -U50,488 and morphine and their reversal with norbinaltorphimine, or with naltrexone and methyl-naltrexone, respectively.

Results: Cervical distension produced a stimulus-dependent increase in electromyographic activity, with a threshold of 25 g. Morphine and -U50,488 produced dose-dependent inhibition of the reflex activity. Log linear regression analysis demonstrated an ID50 of 0.03 for morphine, and of 0.05 mg/kg for -U50,488. These effects were reversed by naltrexone, but not by methylnaltrexone or norbinaltorphimine.     

RELATIONSHIP BETWEEN ANALGESIA AND RESPIRATORY DEPRESSION FOR MU OPIOID RECEPTOR AGONISTS IN MICE

The relationship between analgesic activity, measured as thehot plate reaction time, and respiratory depression, measuredas ventilatory frequency, was investigated in mice for a varietyof mu opioid receptor agonists with differing selectivitiesfor mu receptors compared with delta receptors. There was aweak correlation between analgesia and respiratory depressionfor opioids with the greatest selectivity for mu opioid receptorscompared with delta receptors, such as alfentanil. The strengthof the correlation increased for opioids which had greater deltareceptor activity, such as morphine and fentanyl. Etorphine,which has almost equal affinity for mu, delta and, incidentally,kappa receptors, showed a strong correlation between analgesiaand respiratory depression. We conclude that the predictabilityof the degree of respiratory depression produced by a givenanalgesic dose of an opioid appears to decrease with its selectivityfor mu opioid receptors, at least in the mouse.     

κ-Opioid Receptors Mediate Cardioprotection by Remote Preconditioning

Background: Remote preconditioning is known to be cardioprotective, but the exact mechanism has not been fully elucidated. The objective of the current study was to investigate the role of [kappa]-opioid receptors in cardioprotection by remote preconditioning and reveal possible underlying mechanisms.

Methods: Remote preconditioning was induced in anesthetized male Sprague-Dawley rats by three cycles of 5 min of right femoral artery occlusion followed by 5 min of reperfusion. Myocardial ischemia-reperfusion was achieved by ligation of the left anterior descending coronary artery for 30 min and then reperfusion for 120 min. Infarct size was determined by 2,3,5-triphenyltetrazolium chloride staining. Levels of lactate dehydrogenase, dynorphin, and met-enkephalin in plasma were measured. The opening of the mitochondrial permeability transition pore was monitored with fluorescent calcein in isolated ventricular myocytes.

Results: Both remote preconditioning and U-50,488H (10 mg/kg intravenous), a [kappa]-opioid receptor agonist, significantly decreased the infarct size and plasma lactate dehydrogenase level induced by ischemia-reperfusion, and these effects were attenuated by nor-binaltorphimine (10 mg/kg intravenous), a [kappa]-opioid receptor antagonist, and atractyloside (5 mg/kg intravenous), a mitochondrial permeability transition pore activator. However, administration of naltrindole (5 mg/kg), a [delta]-opioid receptor antagonist, had no effect on the cardioprotection by remote preconditioning. The dynorphin plasma level was increased after remote preconditioning treatment, but the met-enkephalin level did not change. In isolated ventricular myocytes loaded with calcein, U-50,488H (300 [mu]m) decreased the mitochondrial permeability transition pore opening induced by calcium (200 [mu]m), and this effect was attenuated by cotreatment with nor-binaltorphimine (5 [mu]m) or atractyloside (20 [mu]m).     

Supraspinal Antinociceptive Effects of μ and δ Agonists Involve Modulation of Adenosine Uptake

Background: The modulation of extracellular adenosine concentration by opioids provides evidence that the antinociceptive effects of these compounds involve endogenous adenosine. The aim of this study was to determine whether there is a relation between the inhibition of brain synaptosome adenosine uptake by opioid agonists and the analgesic effects of these compounds.

Methods: The authors used the hot plate and tail-pinch tests to evaluate in mice (C57BL/6 females; weight, 25-30 g) the effects of caffeine, a nonspecific adenosine receptor antagonist, on the antinociceptive effect induced by the intracerebroventricular administration of oxymorphone as a [mu] agonist, SNC80 as a [delta] agonist, or U69593 as a [kappa] agonist. They also investigated the effect of these opioid receptor agonists on the uptake of adenosine by whole brain synaptosomes.

Results: Caffeine decreased the analgesic effects induced by oxymorphone or SNC80 but not those induced by U69593. Oxymorphone and SNC80 inhibited adenosine uptake by brain cells, but U69593 did not.     

Ketamine Inhibits Inspiratory-evoked γ-Aminobutyric Acid and Glycine Neurotransmission to Cardiac Vagal Neurons in the Nucleus Ambiguus

Background: Ketamine can be used for perioperative pain management as well as a dissociative anesthetic agent in emergency situations. However, ketamine can induce both cardiovascular and respiratory depression, especially in pediatric patients. Although ketamine has usually been regarded as sympathoexcitatory, recent work has demonstrated that ketamine has important actions on parasympathetic cardiac vagal efferent activity. The current study tests the hypothesis that ketamine, at clinical relevant concentrations, alters central cardiorespiratory interactions in the brainstem and, in particular, the inspiration-evoked increase in [gamma]-aminobutyric acid-mediated and glycinergic neurotransmission to parasympathetic cardiac efferent neurons.

Methods: Cardiac vagal neurons were identified by the presence of a retrograde fluorescent tracer. Respiratory evoked [gamma]-aminobutyric acid-mediated and glycinergic synaptic currents were recorded in cardiac vagal neurons using whole cell patch clamp techniques while spontaneous rhythmic respiratory activity was recorded simultaneously.

Results: Ketamine, at concentrations from 0.1 to 10 [mu]m, evoked a concentration-dependent inhibition of inspiratory burst frequency. Inspiration-evoked [gamma]-aminobutyric acid-mediated neurotransmission to cardiac vagal neurons was inhibited at ketamine concentrations of 0.5 and 1 [mu]m. The increase in glycinergic activity to cardiac vagal neurons during inspiration was also inhibited at ketamine concentrations of 0.5 and 1 [mu]m.     

Enflurane Directly Depresses Glutamate AMPA and NMDA Currents in Mouse Spinal Cord Motor Neurons Independent of Actions on GABAA or Glycine Receptors

Background: The spinal cord is an important anatomic site at which volatile agents act to prevent movement in response to a noxious stimulus. This study was designed to test the hypothesis that enflurane acts directly on motor neurons to inhibit excitatory synaptic transmission at glutamate receptors.

Methods: Whole-cell recordings were made in visually identified motor neurons in spinal cord slices from 1- to 4-day-old mice. Excitatory postsynaptic currents (EPSCs) or potentials (EPSPs) were evoked by electrical stimulation of the dorsal root entry area or dorsal horn. The EPSCs were isolated pharmacologically into glutamate N-methyl-d-aspartate (NMDA) receptor- and non-NMDA receptor-mediated components by using selective antagonists. Currents also were evoked by brief pulse pressure ejection of glutamate under various conditions of pharmacologic blockade. Enflurane was made up as a saturated stock solution and diluted in the superfusate; concentrations were measured using gas chromatography.

Results: Excitatory postsynaptic currents and EPSPs recorded from motor neurons by stimulation in the dorsal horn were mediated by glutamate receptors of both non-NMDA and NMDA subtypes. Enflurane at a general anesthetic concentration (one minimum alveolar anesthetic concentration) reversibly depressed EPSCs and EPSPs. Enflurane also depressed glutamate-evoked currents in the presence of tetrodotoxin (300 nm), showing that its actions are postsynaptic. Block of inhibitory [gamma]-aminobutyric acid A and glycine receptors by bicuculline (20 [mu]m) or strychnine (2 [mu]m) or both did not significantly reduce the effects of enflurane on glutamate-evoked currents. Enflurane also depressed glutamate-evoked currents if the inhibitory receptors were blocked and if either D,L-2-amino-5-phosphonopentanoic acid (50 [mu]m) or 6-cyano-7-nitroquinoxaline-2,3-dione disodium (10 [mu]m) was applied to block NMDA or [alpha]-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-kainate receptors respectively.