Antinociceptive action of epidural K+(ATP) channel openers via interaction with morphine and an alpha(2)- adrenergic agonist in rats

Potassium (K(+)) channels may play some role in the analgesic actions of mu-opioid agonists and alpha(2)-adrenergic agonists (alpha(2) agonists). We examined whether the adenosine triphosphate-sensitive K(+)(K(+)(ATP)) channel openers, levcromakalim and nicorandil, (given epidurally), might have antinociceptive effects in a tail flick test in adult male Sprague-Dawley rats implanted with a lumbar epidural catheter. The interactions with morphine and an alpha(2) agonist were also examined. The epidural administration of levcromakalim (10 microg, 100 microg) or nicorandil (10 microg, 100 microg) alone did not produce antinociception, but 100 microg levcromakalim or nicorandil did potentiate the antinociceptive effect induced by epidural morphine. Epidural glibenclamide (10 microg), a K(+)(ATP) channel blocker, or naloxone (10 microg) antagonized this potentiation. Systemic administration of levcromakalim or nicorandil (at the same dose as that given into the epidural space) did not potentiate the epidural morphine-induced analgesia. A combination of epidural dexmedetomidine (1 microg) and morphine (1 microg) (each at a subantinociceptive dose) had a significant antinociceptive effect, and epidural glibenclamide (10 microg) partly antagonized this antinociception. These data suggest that levcromakalim and nicorandil potentiate the analgesic action of both morphine and dexmedetomidine, probably via an activation of K(+)(ATP) channels at the spinal cord level.     

N-Methyl-D-aspartate receptor antagonist MK-801 attenuates morphine tolerance and associated glial fibrillary acid protein up-regulation: a proteomic approach

Background: It is well known that long-term morphine administration results in tolerance, which limits the clinical use of this drug in pain management.
Methods: Male Wistar rats were randomly assigned to receive one of four different infusions: morphine [15 μg/h, intrathecal (i.t.)], saline, MK-801 (5 μg/h, i.t.) plus morphine (15 μg/h, i.t.), or MK-801 (5 μg/h, i.t.) alone.
Results: Morphine infusion induced a maximal antinociceptive effect on day 1 and tolerance on day 3, and the maximal anti-receptive tolerance was observed on day 5. Co-infusing MK-801 with morphine attenuated morphine's anti-receptive tolerance. Two-dimensional gel electrophoretic analysis of spinal proteins revealed that eight protein spots were up-regulated in morphine-tolerant rats, and that they were significantly inhibited by MK-801 co-infusion. Among the up-regulated proteins, glial fibrillary acid protein (GFAP), a glial-specific maker, was identified by mass spectrometry. This finding was also confirmed by Western blot analysis.
Conclusion: Using proteomic analysis, we identified eight GFAP protein spots that were up-regulated in the dorsal horn of morphine-tolerant rat spinal cords. This up-regulation was partly inhibited by N -methyl- d -aspartate receptor antagonist MK-801 co-infusion, which suggests that GFAP protein can be considered to be a pathogenesis marker of morphine tolerance.     

Antinociceptive interactions between alpha 2-adrenergic and opiate agonists at the spinal level in rodents

This study was undertaken to evaluate the antinociceptive interactions of alpha 2 adrenergic and opiate receptors at the spinal level. Morphine and clonidine were administered intrathecally (i.t.) by lumbar puncture to rats either alone or in the presence of either i.t. yohimbine, an alpha 2 antagonist, or systemic naloxone, an opioid antagonist. The effect of tolerance to systematically administered morphine on responses to i.t. morphine and clonidine was examined in mice. Antinociception was determined by observing the response to a clamp applied to the tail (Haffner test) in mice and by the tail-flick test in rats; log dose-response curves for antinociception were generated for morphine, clonidine, and each drug combination. Morphine and clonidine both produced dose-dependent antinociception when given i.t. in both species. The i.t. administration of yohimbine attenuated the antinociceptive effect of both clonidine and morphine, but naloxone attenuated only the response to morphine. Further, a sub-analgetic dose of i.t. clonidine potentiated the effect of i.t. morphine. In morphine-tolerant mice, i.t. morphine was not efficacious whereas clonidine retained full efficacy, although potency was slightly diminished. Thus, it appears that alpha 2 adrenoceptor-mediated antinociception is independent of opiate receptor mechanisms. Clinical use of intrathecal combinations of alpha 2 adrenergic and opiate receptor agonists to increase analgesia and use of intrathecal alpha 2 agonists for pain relief in patients tolerant to opiates might deserve evaluation.     

Spinal endogenous acetylcholine contributes to the analgesic effect of systemic morphine in rats

BACKGROUND: Systemic morphine is known to cause increased release of acetyicholine in the spinal cord. Intrathecal injection of the cholinergic receptor agonists or acetyicholinesterase inhibitors produces antinociception in both animals and humans. In the present study, we explored the functional importance of spinal endogenous acetylcholine in the analgesic action produced by intravenous morphine. METHODS: Rats were implanted with intravenous and intrathecal catheters. The antinociceptive effect of morphine was determined by the paw-withdrawal latency in response to a radiant heat stimulus after intrathecal treatment with atropine (a muscarinic receptor antagonist), mecamylamine (a nicotinic receptor antagonist), or cholinergic neurotoxins (ethylcholine mustard aziridinium ion [AF64A] and hemicholinium-3). RESULTS: Intravenous injection of 2.5 mg/kg morphine increased significantly the paw-withdrawal latency. Intrathecal pretreatment with 30 microg atropine (n = 7) or 50 microg mecamylamine (n = 6) both attenuated significantly the antinociceptive effect of morphine. The inhibitory effect of atropine on the effect of morphine was greater than that of mecamylanilne. Furthermore, the antinociceptive effect of morphine was significantly reduced in rats pretreated with intrathecal AF64A (n = 7) or hemicholinium-3 (n = 6) to inhibit the high-affinity choline transporter and acetylcholine synthesis. We found that intrathecal AF64A reduced significantly the [3H]hemicholinium-3 binding sites but did not affect its affinity in the dorsal spinal cord. CONCLUSIONS: The data in the current study indicate that spinal endogenous acetylcholine plays an important role in mediating the analgesic effect of systemic morphine through both muscarinic and nicotinic receptors.     

Intracerebroventricular morphine produces antinociception by evoking gamma-aminobutyric acid release through activation of 5-hydroxytryptamine 3 receptors in the spinal cord

BACKGROUND: It has been generally considered that supraspinal morphine activates the serotonergic descending inhibitory system and releases serotonin (5-hydroxytryptamine [5-HT]) in the spinal cord, producing antinociception through activation of 5-HT receptors. The involvement of a spinal gamma-aminobutyric acid-mediated (GABAergic) system is also suggested in supraspinal morphine antinociception. It has been reported that spinal GABAergic system contributes to 5-HT3 receptor-mediated antinociception. In this study, the authors investigated the contribution of spinal 5-HT3 receptor and the GABAergic system in the intracerebroventricular morphine-induced antinociception. METHODS: Male Sprague-Dawley rats were used. Using the spinal microdialysis method, concentrations of 5-HT and GABA were measured after intracerebroventricular morphine administration. The effect of intracerebroventricular naloxone or spinal perfusion of a selective 5-HT3 receptor antagonist 3-tropanyl-indole-3-carboxylate methiodide on the spinal release of GABA after intracerebroventricular morphine administration was also examined. In the behavioral study, involvement of 5-HT3 receptors or GABAA receptors in the intracerebroventricular morphine-induced antinociceptive effect was investigated using the tail-flick test. RESULTS: Intracerebroventricular morphine (40 nmol) significantly increased spinal GABA and 5-HT release. Evoked spinal GABA release was reversed by intracerebroventricular naloxone (40 nmol) or spinal perfusion of 3-tropanyl-indole-3-carboxylate methiodide (1 mm). In the behavioral study, intracerebroventricular morphine produced significant antinociception. Intrathecal administration of either GABAA receptor antagonist bicuculine or 3-tropanyl-indole-3-carboxylate methiodide but not vehicle reversed the morphine-induced antinociceptive effect. CONCLUSION: Intracerebroventricular morphine evokes spinal GABA release via the activation of 5-HT3 receptors in the spinal cord, resulting in antinociceptive effect.     

Spinal glucocorticoid receptors contribute to the development of morphine tolerance in rats

BACKGROUND: Opioid analgesic tolerance is a pharmacologic phenomenon involving the mechanisms of cellular adaptation. Central glucocorticoid receptors (GRs) have been implicated in the cellular mechanism of neuronal plasticity that has many cellular steps in common with the mechanism of opioid tolerance. In a rat model of morphine tolerance, the authors examined the hypothesis that spinal GRs would play a significant role in the development of tolerance to the antinociceptive effect of morphine. METHODS: In experiment 1, each group of rats received the GR antagonist RU38486 (0.5 or 1 microg), the mineralocorticoid receptor antagonist spironolactone (3 microg), or a vehicle, given intrathecally with morphine (10 microg) twice daily for 6 days. In experiment 2, four groups of rats were used, and each group received intrathecally 10 microg morphine plus 5 micromol GR antisense oligodeoxynucleotide, sense oligodeoxynucleotide, mixed-base oligodeoxynucleotide, or vehicle. Western blotting was used to examine the expression of GRs within the spinal cord dorsal horn. In experiment 3, the GR agonist dexamethasone (4 microg) was given intrathecally twice daily in combination with 10 microg morphine. For all experiments, the development of morphine antinociceptive tolerance was assessed using the tail-flick test. RESULTS: The development of tolerance to the antinociceptive effect of morphine was substantially attenuated when the GR antagonist RU38486 (1 > 0.5 microg > vehicle) but not spironolactone was coadministered with morphine for 6 days. A single treatment with RU38486 did not affect morphine antinociception, nor did it reverse morphine tolerance on day 7. A similar reduction of morphine tolerance was observed in those rats treated with a GR antisense oligodeoxynucleotide but not a sense or mixed-base oligodeoxynucleotide. The administration of the GR antisense oligodeoxynucleotide also prevented GR up-regulation within the spinal cord dorsal horn. Moreover, the GR agonist dexamethasone facilitated the development of morphine tolerance. CONCLUSIONS: The results indicate an important role of spinal GRs in the cellular mechanisms of morphine tolerance in rats and may have significant implications in clinical opioid therapy.     

The effects of intrathecal morphine encapsulated in L- and D-dipalmitoylphosphatidyl choline liposomes on acute nociception in rats

Liposomes can serve as a sustained-release carrier system, permitting the spinal delivery of large opioid doses restricting the dose for acute systemic uptake. We evaluated the antinociceptive effects of morphine encapsulated in liposomes of two isomeric phospholipids, L-dipalmitoylphosphatidyl choline (L-DPPC) and D-dipalmitoylphosphatidyl choline (D-DPPC), in comparison with morphine in saline. Sprague-Dawley rats with chronic lumbar intrathecal catheters were tested for their acute nociceptive response using a hindpaw thermal escape test. Their general behavior, motor function, pinna reflex, and corneal reflex were also examined. The duration of antinociception was longer in both liposomal morphine groups than in the free morphine group. The peak antinociceptive effects were observed within 30 min after intrathecal morphine, L-DPPC or D-DPPC morphine injection. The rank order of the area under the effect-time curve for antinociception was L-DPPC morphine > D-DPPC morphine > morphine. The 50% effective dose was: 2.7 microg (morphine), 4.6 microg (L-DPPC morphine), and 6.4 microg (D-DPPC morphine). D-DPPC morphine had less side effects for a given antinociceptive AUC than morphine. In conclusion, L-DPPC and D-DPPC liposome encapsulation of morphine prolonged the antinociceptive effect on acute thermal stimulation and could decrease side effects, compared with morphine alone. Implications: Two isomers of liposome (L-dipalmitoylphosphatidyl choline and D-dipalmitoylphosphatidyl choline) encapsulation of morphine prolonged the analgesic effect on acute thermal-induced pain when administered intrathecally and could decrease side effects, compared with morphine alone.     

Morphine tolerance increases [3H]MK-801 binding affinity and constitutive neuronal nitric oxide synthase expression in rat spinal cord

N-Methyl-D-aspartate (NMDA) receptor antagonists and nitricoxide synthase (NOS) inhibitors inhibit morphine tolerance.In the present study, a lumbar subarachnoid polyethylene (PE₁₀)catheter was implanted for drug administration to study alterationsin NMDA receptor activity and NOS protein expression in a morphine-tolerantrat spinal model. Antinociceptive tolerance was induced by intrathecal(i.t.) morphine infusion (10 µg h^–1) for 5 days.Co-administered (+)-5-methyl-10,11-dihydro-⁵H-dibenzo[a,d]cyclohepten-5,10-iminemaleate (MK-801) (10 µg h^–1 i.t.) with morphinewas used to inhibit the development of morphine tolerance. Lumbarspinal cord segments were removed and prepared for [³H]MK-801binding assays and NOS western blotting. The binding affinityof [³H]MK-801 was higher in spinal cords of morphine-tolerantrats (mean (SEM) K_D=0.41 (0.09) nM) than in control rats (1.50(0.13) nM). There was no difference in B_max. Western blot analysisshowed that constitutive expression of neuronal NOS (nNOS) proteinin the morphine-tolerant group was twice that in the controlgroup. This up-regulation was partially prevented by MK-801.The results suggest that morphine tolerance affects NMDA receptorbinding activity and increases nNOS expression in the rat spinalcord. Br J Anaesth 2000; 85: 587–91 ^* Corresponding author     

Thermal and mechanical antinociceptive action of spinal vs peripherally administered clonidine in the rat inflamed knee joint model

It has been demonstrated recently that in addition to its spinal analgesic actions, the alpha 2 adrenoreceptor agonist clonidine also has peripheral analgesic activity. Few data are available regarding the antinociceptive effects of spinal vs peripherally delivered clonidine in inflammatory pain. Thus we have studied spinal (intrathecal = i.t.) and peripheral (intra-articular = i.a.) administration of clonidine in the rat inflamed knee joint model. Thermal and mechanical antinociception was assessed in rats over 28 h using a modified Hargreaves box and von Frey hairs after induction of tonic persistent inflammatory pain by injection of a kaolin-carrageenan mixture into the right knee joint. Thirty minutes after injection of kaolin-carrageenan, clonidine was administered via an i.t. catheter or by i.a. injection into the right inflamed knee joint or by subcutaneous injection (s.c.) (highest effective intra-articular dose). The specific site of action was assessed using the alpha 2 antagonist yohimbine i.t., i.a. or s.c. Clonidine i.t. resulted in thermal and mechanical antinociception during ongoing inflammation, which was not enhanced by inflammation. In contrast, i.a. delivery of clonidine, which also produced a dose- dependent thermal and mechanical antinociceptive effect, revealed a leftward shift in the antinociceptive activity produced by ongoing inflammation. Yohimbine inhibited the antinociceptive action of clonidine at the site of delivery. We suggest that clonidine produces potent thermal and mechanical antinociception regardless of the route of administration. However, chronic inflammatory processing appears to enhance the antinociceptive efficacy of the peripheral alpha 2 agonist.      

Experimental arthritis in the rat does not alter the analgesic potency of intrathecal or intraarticular morphine.

To explore further the role of inflammatory processing on peripheral opioid pharmacology, we examined whether the potency of intraarticular (i.a.) or intrathecal (i.t) morphine in tests of thermal and mechanical nociception changed during the induction of experimental arthritis in the rat. Thermal nociception by i.t. morphine (3, 10, and 50 micrograms) or i.a. morphine (100, 1000, and 3000 micrograms) was assessed by means of a modified Hargreaves box ever) 28 h. Mechanical antinociception was determined for the largest applied doses of morphine using von Frey hairs. Morphine produced dose-dependent thermal antinociception after i.t. or i.a. administration: a 50% increase in maximum antinociceptive thermal response (50% effective dose) was produced by i.t. doses of 9.7 micrograms at the start and 9.1 micrograms at the end of this 28-h observational interval, whereas after i.a. administration, 50% effective dose values were 553 micrograms at the start and 660 micrograms at the end. The largest applied dose of either i.t. or i.a. morphine produced mechanical antinociception. On Day 1, the antinociceptive effect for mechanical nociception (expressed as the area under the curve of the percentage of maximal possible effect values at 0.5, 1, 2, and 4 h) was 68% for i.t. morphine 50 micrograms and 53% for i.a. morphine 3000 micrograms. Neither result differed from the corresponding area under the curve values on Day 2. Naloxone administered either i.t. or i.a. abolished the antinociceptive action of morphine given at the same site. We conclude that, although morphine has a peripheral analgesic site of action in a rat arthritis model, its potency for both i.a. and i.t. routes of administration does not change during the onset of arthritis. Implications: In this animal study, we showed that the administration of morphine modulates thermal and mechanical antinociception at central and peripheral sites in inflammatory pain.     

Differential effects of intrathecal midazolam on morphine-induced antinociception in the rat: role of spinal opioid receptors.

The antinociceptive effects of an intrathecally administered benzodiazepine agonist midazolam, alone and in combination with morphine, were examined in the rat by using the tail-flick test. The duration of antinociceptive effect produced by midazolam was significantly less (P less than 0.05) than that produced by morphine. Low doses of midazolam (10 micrograms) and morphine (10 micrograms) produced a synergistic effect in prolonging antinociceptive effect. However, at higher doses (20 or 30 micrograms), these drugs reduced the extent of antinociception produced by each other. Naloxone administration prevented antinociception produced by these drugs, indicating interactions between midazolam and opioid receptors. Midazolam had dual effects on the binding of opioid ligands to the spinal opioid receptors. At low dose, it potentiated the displacement of [3H]naloxone by morphine. At higher doses, midazolam inhibited the binding of opioid ligands to their spinal receptors in the following order: kappa greater than delta greater than mu. These results indicate that differential antinociceptive effects of midazolam on morphine-induced antinociception involve interaction of this benzodiazepine with spinal opioid receptors.     

Antinociceptive effect of riluzole in rats with neuropathic spinal cord injury pain

Symptoms of neuropathic spinal cord injury (SCI) pain include cutaneous hypersensitivity and spontaneous pain below the level of the injury. Riluzole, an FDA-approved drug for the treatment of amyotrophic lateral sclerosis, has been demonstrated to attenuate neural excitotoxicity by blocking the effects of the excitatory amino acid glutamate on glutamate receptors and by inhibiting voltage-gated Na(+) and Ca(2+) channels. Neuropathic pain in rat models of SCI is thought to be mediated by dysfunctional ion channels and glutamate receptors expressed on CNS neurons. Thus riluzole's mechanism of action could be relevant in treating neuropathic SCI pain. The current study evaluated the antinociceptive potential of riluzole in rats following a SCI. Four weeks after a brief compressive injury to the mid-thoracic spinal cord, rats displayed significantly decreased hind paw withdrawal thresholds, suggestive of below-level cutaneous hypersensitivity. A single systemic dose of riluzole (8?mg/kg) injected intraperitoneally (i.p.) reversed cutaneous hypersensitivity in SCI rats. To identify riluzole's CNS site of action, riluzole was injected intrathecally (i.t.) and intracerebroventricularly (i.c.v.) in SCI rats. Significant antinociceptive effects were obtained following i.c.v., but not i.t., injection. Systemic riluzole was also antinociceptive in uninjured rats, increasing the latency to respond to an acute noxious thermal stimulus in the tail flick test. Unlike in SCI rats, however, riluzole was not effective when administered directly into the CNS, indicating a peripherally mediated antinociceptive mechanism. Although riluzole appears to have a general antinociceptive effect, the site of action may be model dependent. In total, these data indicate that riluzole may be an effective clinical analgesic for the treatment of below-level neuropathic SCI pain. Although the exact mechanism of action is not clear, there is a predominant supraspinal component of riluzole-induced antinociception in SCI rats.     

Intracerebroventricular Morphine Produces Antinociception by Evoking γ-Aminobutyric Acid Release through Activation of 5-Hydroxytryptamine 3 Receptors in the Spinal Cord

Background: It has been generally considered that supraspinal morphine activates the serotonergic descending inhibitory system and releases serotonin (5-hydroxytryptamine [5-HT]) in the spinal cord, producing antinociception through activation of 5-HT receptors. The involvement of a spinal [gamma]-aminobutyric acid-mediated (GABAergic) system is also suggested in supraspinal morphine antinociception. It has been reported that spinal GABAergic system contributes to 5-HT3 receptor-mediated antinociception. In this study, the authors investigated the contribution of spinal 5-HT3 receptor and the GABAergic system in the intracerebroventricular morphine-induced antinociception.

Methods: Male Sprague-Dawley rats were used. Using the spinal microdialysis method, concentrations of 5-HT and GABA were measured after intracerebroventricular morphine administration. The effect of intracerebroventricular naloxone or spinal perfusion of a selective 5-HT3 receptor antagonist 3-tropanyl-indole-3-carboxylate methiodide on the spinal release of GABA after intracerebroventricular morphine administration was also examined. In the behavioral study, involvement of 5-HT3 receptors or GABAA receptors in the intracerebroventricular morphine-induced antinociceptive effect was investigated using the tail-flick test.

Results: Intracerebroventricular morphine (40 nmol) significantly increased spinal GABA and 5-HT release. Evoked spinal GABA release was reversed by intracerebroventricular naloxone (40 nmol) or spinal perfusion of 3-tropanyl-indole-3-carboxylate methiodide (1 mm). In the behavioral study, intracerebroventricular morphine produced significant antinociception. Intrathecal administration of either GABAA receptor antagonist bicuculine or 3-tropanyl-indole-3-carboxylate methiodide but not vehicle reversed the morphine-induced antinociceptive effect.     

Spinally mediated antinociception following intrathecal chlordiazepoxide--further evidence for a benzodiazepine spinal analgesic effect.

This is a report of the results of 25 experiments in five rats investigating the dose-response relationship for the antinociceptive effects of chlordiazepoxide given intrathecally in the dose range 0.03-0.9 mumol and a further 40 experiments in eight rats investigating the actions of flumazenil and naloxone on this effect. Electrical-current thresholds for pain were measured in the skin of the tail and neck of rats with previously implanted lumbar subarachnoid catheters. Intrathecal chlordiazepoxide produced spinally mediated antinociception, i.e. rises in the current threshold for pain in the tail without a significant change in the neck. This antinociceptive effect was dose dependent. Flumazenil 16.5 mumol kg-1 i.p. reduced the response caused by chlordiazepoxide 0.6 mumol by 78 +/- 6% (mean +/- SEM). By contrast, the same dose of flumazenil did not significantly affect the antinociceptive effect of an equipotent dose of intrathecal fentanyl 0.74 nmol. Naloxone 0.38 mumol kg-1 i.p. abolished the spinally mediated antinociception caused by fentanyl (96 +/- 7% suppression) but did not significantly reduce the effect of chlordiazepoxide (27 +/- 13% suppression). However, a higher dose of naloxone (6.1 mumol kg-1 i.p.) caused significant partial suppression (79 +/- 10.7%) of chlordiazepoxide spinal antinociception. We conclude that chlordiazepoxide produces an antinociceptive effect by combination with benzodiazepine receptors in the spinal cord.     

The activation of spinal N-methyl-D-aspartate receptors may contribute to degeneration of spinal motor neurons induced by neuraxial morphine after a noninjurious interval of spinal cord ischemia

We investigated the relationship between the degeneration of spinal motor neurons and activation of N-methyl-d-aspartate (NMDA) receptors after neuraxial morphine following a noninjurious interval of aortic occlusion in rats. Spinal cord ischemia was induced by aortic occlusion for 6 min with a balloon catheter. In a microdialysis study, 10 muL of saline (group C; n = 8) or 30 mug of morphine (group M; n = 8) was injected intrathecally (IT) 0.5 h after reflow, and 30 mug of morphine (group SM; n = 8) or 10 muL of saline (group SC; n = 8) was injected IT 0.5 h after sham operation. Microdialysis samples were collected preischemia, before IT injection, and at 2, 4, 8, 24, and 48 h of reperfusion (after IT injection). Second, we investigated the effect of IT MK-801 (30 mug) on the histopathologic changes in the spinal cord after morphine-induced spastic paraparesis. After IT morphine, the cerebrospinal fluid (CSF) glutamate concentration was increased in group M relative to both baseline and group C (P < 0.05). This increase persisted for 8 hrs. IT MK-801 significantly reduced the number of dark-stained alpha-motoneurons after morphine-induced spastic paraparesis compared with the saline group. These data indicate that IT morphine induces spastic paraparesis with a concomitant increase in CSF glutamate, which is involved in NMDA receptor activation. We suggest that opioids may be neurotoxic in the setting of spinal cord ischemia via NMDA receptor activation.     

Sedative but not analgesic alpha2 agonist tolerance is blocked by NMDA receptor and nitric oxide synthase inhibitors

BACKGROUND: Studies show that the sedative and analgesic effects of alpha2 adrenergic agonists decrease over time, which is a form of synaptic plasticity referred to as tolerance. Because both the N-methyl-D-aspartate (NMDA) receptor complex and nitric oxide synthase are pivotal for some forms of synaptic plasticity, their role in tolerance to the hypnotic and analgesic effects of alpha2 agonists was investigated. METHODS: After institutional approval, rats were made tolerant to the hypnotic or analgesic action of an alpha2 agonist, dexmedetomidine. The hypnotic response to dexmedetomidine was assessed by the duration of loss of righting reflex, and the analgesic response to dexmedetomidine was assessed by the tail-flick assay. In separate cohorts, either the NMDA receptors or nitric oxide synthase was antagonized by coadministration of MK-801, ketamine, or NO2-arginine, respectively, during induction of tolerance. In a separate series of experiments, after tolerance was induced, the hypnotic and analgesic responses to dexmedetomidine were assessed in the presence of acutely administered MK-801 or NO2-arginine. RESULTS: Induction of tolerance to the hypnotic effect of dexmedetomidine is blocked by coadministration of MK-801, ketamine, and NO2-arginine. However, after tolerance developed, acute administration of MK-801, ketamine, or NO2-arginine did not prevent the expression of tolerance. Coadministration of MK-801 or NO2-arginine neither prevents the development nor reverses the expression of tolerance to the analgesic action of dexmedetomidine. CONCLUSION: The underlying processes responsible for the development of tolerance to the hypnotic and analgesic actions of systemically administered alpha2 agonists were different, with only the sedative tolerance involving the NMDA receptor and nitric oxide synthase system.     

The antinociceptive effect of the combination of spinal morphine with systemic morphine or buprenorphine.

We sought to analyze the mode of interaction of spinal morphine with systemic morphine or buprenorphine, administered in a wide range of antinociceptive doses. The study was performed on Sprague-Dawley rats by using a plantar stimulation test and isobolographic and fractional analyses of drug interaction. The isobolographic and fractional analyses demonstrated that intrathecal morphine interacted with subcutaneous morphine in a synergistic manner while producing a 50% or 75% antinociceptive effect. The sum of D(75) fractions was more than that for 50% antinociception, suggesting a less dramatic interaction. The combination with a maximal relative dose of systemic morphine (0.66:1) showed a maximal degree of supraadditivity. The interaction between spinal morphine and systemic buprenorphine was similar to that of morphine/morphine, although the supra-additivity was not as pronounced. For the doses that produced a 50% antinociceptive effect, a synergistic interaction was observed only for the combination with a morphine/buprenorphine ratio of 1.33:1. When the relative amount of intrathecal morphine was decreased or increased, the effect became additive. At the doses that produced 75% antinociception, both combinations of morphine and buprenorphine demonstrated supraadditive interaction. Implications: Spinal morphine interacts with systemic morphine or buprenorphine in asupraadditive manner. This mode of interaction most probably results from the simultaneous activation of spinal and supraspinal antinociceptive systems.Supraspinal structures played a more important role in the antinociceptive effect of experimental combinations than structures of the spinal cord.     

nPKCepsilon and NMDA receptors participate in neuroprotection induced by morphine pretreatment

Morphine pretreatment induces ischemic tolerance in neurons, but it remains uncertain whether novel protein kinase C epsilon isoform (nPKCepsilon) and N-methyl-D-aspartate (NMDA) receptors are involved in this neuroprotection. The present study examined this issue. Hippocampal slices from adult BALB/C mice were incubated with morphine at 0.1-10.0 muM in the presence or absence of various antagonists for 30 minutes and then kept in morphine- and antagonist-free buffer for 30 minutes before being subjected to oxygen-glucose deprivation for 20 minutes. After recovery in oxygenated artificial fluid for 5 hours, assessment of slice injury was done by determination of the intensity of slice stain after they were incubated with 2% 2,3,5-triphenyltetrazolium chloride for 30 minutes and extracted by organic solvent for 24 hours. At designated periods, slices were preserved for immunoblot analysis to observe effects of morphine pretreatment on membrane translocation and total protein expression of nPKCepsilon and phosphorylation of NR1 subunits of NMDA receptors. The neuroprotection induced by morphine pretreatment was partially blocked by chelerythrine (a nonselective PKC blocker), epsilonv(1-2) (a selective nPKCepsilon antagonist), MK-801 (a noncompetitive NMDA receptor blocker), chelerythrine combined with MK-801, and epsilonv(1-2) with MK-801. Morphine pretreatment significantly inhibited nPKCepsilon membrane translocation and phosphorylation of NR1 subunits of NMDA receptors during reperfusion injury. However, epsilonv(1-2) blocked these effects induced by morphine pretreatment. These findings suggested that nPKCepsilon and NMDA receptors might participate in neuroprotection induced by morphine pretreatment, and NMDA receptors might be downstream targets of nPKCepsilon.     

Production of paradoxical sensory hypersensitivity by alpha 2-adrenoreceptor agonists

BACKGROUND: Administration of opioid receptor agonists is followed by paradoxical sensory hypersensitivity. This hypersensitivity has been suggested to contribute to the antinociceptive tolerance observed with opioids. The authors hypothesized that alpha 2-adrenoreceptor agonists, which also produce antinociceptive tolerance, would produce sensory hypersensitivity. METHODS: alpha 2-Adrenoreceptor agonists were administered to male Sprague-Dawley rats as a single subcutaneous injection, a continuous subcutaneous infusion, a single intrathecal injection, or a continuous intrathecal infusion. Thermal sensitivity was determined using latency to withdrawal of the hind paw from radiant heat. Tactile sensitivity was determined using withdrawal threshold to von Frey filaments. Spinal dynorphin content was measured by enzyme immunoassay. RESULTS: Single systemic or intrathecal injections of clonidine or dexmedetomidine produced antinociception followed by delayed thermal and tactile hypersensitivity. Six-day systemic or intrathecal infusion of clonidine produced tactile and thermal hypersensitivity observed even during clonidine infusion. Sensory hypersensitivity was prevented by coadministration of the alpha 2-adrenoreceptor-selective antagonist idazoxan or the N-methyl-D-aspartate receptor-selective antagonist MK-801. Six-day infusion of intrathecal clonidine increased dynorphin content in dorsal lumbar spinal cord. MK-801 and dynorphin antiserum reversed clonidine-induced sensory hypersensitivity. CONCLUSIONS: alpha 2-Adrenoreceptor agonists produce sensory hypersensitivity that may be analogous to that produced by opioids. Sensory hypersensitivity was prevented by idazoxan, demonstrating that it is mediated by alpha 2 receptors. Clonidine infusion increased spinal dynorphin content. Sensory hypersensitivity was prevented or reversed by MK-801 and dynorphin antiserum, implicating N-methyl-D-aspartate receptors and spinal dynorphin in its production. Clinicians should be mindful of the possibility of drug-induced hyperalgesia in patients treated with alpha 2-adrenoreceptor agonists.     

Magnesium sulfate potentiates morphine antinociception at the spinal level

Intrathecal magnesium sulfate coinfusion with morphine increases antinociception in normal rats; however, because magnesium also delays the onset of tolerance, it is not clear whether this additional antinociception is a result of potentiated analgesia or tolerance abatement. We examined the antinociceptive interaction of intrathecal (IT) bolus magnesium sulfate and morphine in morphine naive rats and those with mechanical allodynia after a surgical incision. After intrathecal catheter implantation, rats were given preinjections of magnesium or saline, followed by injections of morphine or saline. In morphine na?ve rats, IT bolus magnesium sulfate 281 and 375 microg followed by IT morphine 0.25 or 0.5 nmol enhanced peak antinociception and area under the response versus time curve two-to-three-fold in the tail-flick test as compared with morphine alone. Likewise, in rats with incisional pain, IT bolus magnesium sulfate 188 and 375 microg followed by morphine 0.5 nmol reduced mechanical allodynia, whereas morphine 0.5 nmol alone did not. This study suggests that IT magnesium sulfate potentiates morphine at a spinal site of action. Implications: Magnesium sulfate potentiates morphine analgesia when coadministered intrathecally in normal rats, and in an animal model of mechanical allodynia after a surgical incision. These results suggest that intrathecal administration of magnesium sulfate may be a useful adjunct to spinal morphine analgesia.