Morphine-3-glucuronide: hyperglycemic and neuroendocrine potentiating effects

The greater potency of morphine-6-glucuronide (M6G) as well as the inactivity of morphine-3-glucuronide (M3G) with respect to the antinociceptive effects of the parent molecule, morphine (MOR), have been well established. It has been suggested that M3G is an antagonist of MOR's antinociceptive and respiratory depressive effects. The present study addressed the central nervous system (CNS) interaction of these opiate metabolites on their metabolic and hormonal effects. Whole body glucose kinetics were assessed on conscious, chronically catheterized, unrestrained rats. M3G (5 μg) or H₂O (5 μl) was injected intracerebroventricularly (i.c.v.) 15 min prior to the bolus administration of H₂O (5 μl), M6G (1 μg), or MOR (80 μg). i.c.v. M3G (5 μg) resulted in behavioral excitation, hyperglycemia (+50%), stimulation of glucose rate of appearance (R_a; +100%), glucose rate of disappeaance (R_d; +70%), and metabolic clearance rate (MCR; +33%) within 30 min after injection with no alterations in hormone concentrations. i.c.v. M6G and MOR produced progressive hyperglycemia with significantly high catecholamine and corticosterone levels. M3G pretreatment resulted in enhanced elevations in plasma glucose levels (+52% and +18%), plasma lactate (+138% and +108%), norepinephrine (+96% and +30%), and epinephrine (+62% and +67%) in response to both i.c.v. MOR and M6G administration. These findings suggest a non-opiate and non-hormonal mechanism for M3G-induced hyperglycemia. In contrast, the metabolic and hormonal responses to i.c.v. M6G and MOR are associated with elevations in catecholamine and corticosterone levels, which are remarkably enhanced by M3G pretreatment, most likely through accelerated catecholamine release. Our findings suggest a modulatory role for MOR glucuronidation, not only by rendering it inactive, as in the case of M3G, but by an interplay of the metabolic effects of the parent molecule and its metabolite     

Central opiate modulation of growth hormone and insulin-like growth factor-I

The effects of central administration of morphine-sulfate (MOR:80 μg) and morphine-6-glucuronide (M6G:1 μg) on the growth hormone (GH)/insulin-like growth factor (IGF) system were assessed. MOR and M6G were injected intracerebroventricularly (ICV) in chronically catheterized 24 h fasted rats; time-matched control animals received H₂O (5 μl). MOR increased plasma GH concentrations 3-fold 2 h after ICV injection, and transiently increased the plasma concentration and liver content of IGF-I (60% and 90%, respectively) 30 min after ICV injection. M6G did not produce any significant alterations in plasma GH and IGF-I levels at the time-points measured. Both MOR and M6G increased the concentration of IGF binding protein-1 (IGFBP-1) in plasma and liver 2 h after injection. However, MOR showed 2- to 2.5-fold greater effect than M6G in stimulating plasma and liver IGFBP-1. MOR and M6G produced similar increases in plasma epinephrine (5-fold), norepinephrine (3-fold) and corticosterone (1.5-fold). Neither opiate significantly altered circulating insulin levels. These findings suggest that opiate modulation of GH and IGF may be hormone-independent and centrally modulated. We speculate that differential affinities of MOR and M6G to the different opiate receptor subtypes might be responsible for their distinct effects on GH/IGF-I system.     

Depression by morphine-6-glucuronide of nociceptive activity in rat thalamus neurons: comparison with morphine

To assess the contribution of the active metabolite of morphine, morphine-6-glucuronide (M6G), to the analgesic effect of systemically administered morphine, experiments were carried out on rats under urethane anesthesia in which nociceptive activity was evoked by electrical stimulation of afferent C fibers in the sural nerve and recorded from single neurons in the ventrobasal complex of the thalamus. Intravenous (i.v.) injections of morphine completely blocked the activity at doses of 500 and 1000 μg/kg, the ED,, being 44 μg/kg. M6G administered by i.v. injection reduced the evoked nociceptive activity only by about 40% at 80 and 160 μg/kg, the ED₅₀ being 6 μg/kg. After intrathecal (i.t.) injection, morphine produced maximum depression of 55% of the control activity at 20 μg the ED₅₀ is 18 μg. M6G injected i.t. produced maximum depression of 40% at doses ranging from 0.2 to 10 μg. The ED₅₀ of M6G i.t. is below 0.2 μg. The effects of morphine and M6G were reversed by naloxone (200 μg/kg i.v.). The results show that M6G is more potent than morphine, regardless of the route of administration, while morphine is more effective when injected i.v. Due to the low efficacy of M6G, it seems unlikely that this glucuronide contributes substantially to the analgesic effect of morphine when renal function is normal. The results also make evident that the maximum effect of morphine results from an action at spinal and supraspinal sites.     

Contribution of excitatory amino acids to morphine-induced metabolic alterations

Previous studies have indicated that excitatory amino acids are involved in the analgesic and addictive properties of morphine. However, their role in the morphine-induced alterations in glucose metabolism is not known. This study assessed the contribution of NMDA receptor activation to the morphine-induced hormonal and metabolic alterations in conscious unrestrained chronically catheterized rats. Whole body glucose flux was assessed with a primed constant intravenous infusion of [3-³H]glucose in rats pretreated with the NMDA-receptor antagonist MK-801 (0.25 mg/kg, intraarterial) or an equal volume (1.5 ml) of sterile saline (0.9% ) administered 15 min prior to i.c.v. injection of H₂O (Con; 5 μl) or morphine sulfate (80 μg). No significant alterations were noted in metabolic and hormonal parameters of H₂O injected rats. i.c.v. morphine increased the plasma glucose concentration (60%), hepatic glucose production (R_a; 60%) and whole body glucose utilization (R_d; 53%), but did not alter the glucose metabolic clearance rate (MCR). MK-801 alone resulted in transient hyperglycemia (25%), stimulation of glucose R_a (60%) and glucose R_d (53%), and a significant (30%) increase in MCR. MK-801 pretreatment blunted the morphine-induced hyperglycemia and the increased glucose R_a and R_d. Morphine increased the plasma concentration of epinephrine (4-fold), norepinephrine (2-fold) and corticosterone (67%); however, no alterations in plasma insulin and glucagon were detected. MK-801 pretreatment, blunted the morphine-induced increase in corticosterone and norepinephrine, and elicited a significant rise in insulin concentrations. These results indicate that activation of the NMDA receptors contributes to the morphine-induced hyperglycemia and hormonal alterations. Furthermore, this response appears partially mediated by activation of sympathetic outflow and suppression of insulin release, which is blunted by inhibition of NMDA receptors.     

The spinal antinociceptive actions of morphine metabolites morphine-6-glucuronide and normorphine in the rat

The profound and prolonged effects of morphine in patients with renal dysfunction have been associated with high plasma levels of the opiate metabolites morphine-6-glucuronide (M6G) and morphine-3-glucuronide (M3G) rather than an increased concentration of morphine. We present here electrophysiological evidence to suggest that potent spinal antinociception can be produced by both M6G and normorphine, another metabolite of morphine. Extracellular recordings of Aβ- and C-fibre-evoked responses of convergent dorsal horn neuroneswere made in the halothane anaesthetised rat. M6G elicited dose-dependent, naloxone-reversible inhibitions of C-fibre-evoked responses which were completely suppressed (8% of control) by 2 μg M6G whereas Aβ-fibre-evoked responses were only reduced to 57% of controls. The ED₅₀ for the effects of M6G on C-fibre-evoked activity was calculated to be 0.53 μg. Systematic administration of M6G (2 mg/kg) also profoundly reduced noxious evoked neuronal activity. intrathecal normorphine was less potent than M6G but complete selective inhibitions of C-fibre-evoked responses could be elicited by 25 μg and the ED₅₀ was calculated to be 2.68 μg. No such inhibitions were observed following administration of M3G. A comparison with intrathecal morphine in the same preparation reveals that normorphine is equipotent with morphine whereas M6G is 13-fold more potent. These results therefore confirm that M6g and normorphine might be significant contributers to opiate analgesia after administration of morphine.     

Differential expression and HIV-1 regulation of μ-opioid receptor splice variants across human central nervous system cell types

The μ-opioid receptor (MOR) is known to undergo extensive alternative splicing as numerous splice variants of MOR have been identified. However, the functional significance of MOR variants, as well as how splice variants other than MOR-1 might differentially regulate human immunodeficiency virus type-1 (HIV-1) pathogenesis in the central nervous system (CNS), or elsewhere, has largely been ignored. Our findings suggest that there are specific differences in the MOR variant expression profile among CNS cell types, and that the expression levels of these variants are differentially regulated by HIV-1. While MOR-1A mRNA was detected in astroglia, microglia, and neurons, MOR-1 and MOR-1X were only found in astroglia. Expression of the various forms of MOR along with the chimeric G protein qi5 in HEK-293T cells resulted in differences in calcium/NFAT signaling with morphine treatment, suggesting that MOR variant expression might underlie functional differences in MOR-effector coupling and intracellular signaling across different cell types. Furthermore, the data suggest that the expression of MOR-1 and other MOR variants may also be differentially regulated in the brains of HIV-infected subjects with varying levels of neurocognitive impairment. Overall, the results reveal an unexpected finding that MOR-1 may not be the predominant form of MOR expressed by some CNS cell types and that other splice variants of MOR-1, with possible differing functions, may contribute to the diversity of MOR-related processes in the CNS.     

Potency ratios of morphine and morphine-6β-glucuronide analgesia elicited from the periaqueductal gray, locus coeruleus or rostral ventromedial medulla of rats

The present study examined whether morphine and morphine-6β-glucuronide (M6G) analgesia on the tail-flick and jump tests differed in potency in the periaqueductal gray, the locus coeruleus or the rostral ventromedial medulla. Morphine and M6G significantly and dose-dependently elicited analgesia on both nociceptive tests from each site. Site-specific differences were observed in the potency of M6G, but not morphine analgesia on both tests. Periaqueductal gray placements displayed analgesic ED_50s on the tail-flick (morphine: 2.1 μg, M6G: 0.2 μg) and jump (morphine: 2.2 μg, M6G: 0.4 μg) tests with respective potency ratios of 12.9 and 6.5. Locus coeruleus placements displayed analgesic ED_50s on the tail-flick (morphine: 1.7 μg, M6G: 0.1 μg) and jump (morphine: 3.4 μg, M6G: 0.2 μg) tests with respective potency ratios of 15.9 and 15.1. Rostral ventromedial placements displayed analgesic ED_50s on the tail-flick (morphine: 1.4 μg, M6G: 0.06 μg) and jump (morphine: 1.9 μg, M6G: 0.08 μg) tests with potency ratios of 21.9 on both tests. The greater analgesic sensitivity of the rostral ventromedial medulla to M6G may be due to either pharmacodynamic (splice variants of the MOR-1 gene) and/or pharmacokinetic (lipid solubility) factors.     

Modification of morphine-induced locomotor activity by pertussis toxin: biochemical and behavioral studies in mice

The effect of pertussis toxin (PTX) on the locomotor-enhancing action of systemic and intracerebroventricular (i.c.v.) morphine was investigated in mice. Mice were i.c.v. injected with either PTX (0.25 and 0.5 μg) or saline as a control. The s.c. (5–20 mg/kg) and i.c.v. (7–30 nmol) administration of morphine produced a dose-related locomotor-enhancing action in control mice. The peak effect of morphine (30 nmol, i.c.v.)-induced hyperlocomotion was observed 90 min after the morphine injection. At the same time, morphine significantly increased dopamine (DA) metabolism in the limbic forebrain (nucleus accumbens and olfactory tubercle). Similarly, the selective μ-opioid receptor agonist[d-Ala²,N-MePhe⁴,Gly-ol⁵]enkephalin (DAGO, 4 nmol, i.c.v.) also significantly increased locomotor activity and DA metabolism in the limbic forebrain. Both morphine- and DAGO-induced hyperlocomotion and elevation of DA turnover were antagonized by pretreatment with the μ antagonist β-funaltrexamine (β-FNA). These results suggest that the locomotor-enhancing action of morphine results from the activation of central μ-opioid receptors, and that the activation of the mesolimbic DA system may be involved in the expression of morphine-induced hyperlocomotion in mice. Furthermore, pretreatment with PTX (0.5 μg, i.c.v., 6 days prior to the testing) significantly reduced hyperlocomotion and elevation of DA turnover in the limbic forebrain which had been induced by administrations of morphine (30 nmol, i.c.v.) and DAGO (4 nmol, i.c.v.). These findings suggest that the central PTX-sensitive GTP-binding protein (G-protein) mechanism may play an important role in opioids-induced locomotor-enhancing action. Furthermore, the activation of mesolimbic DA transmission by μ-opioid agonists may also be mediated by a PTX-sensitive G-protein mechanism in mice.     

Pharmacological characterization of morphine-6-sulfate and codeine-6-sulfate

Morphine-6-sulfate (M6S) and codeine-6-sulfate (C6S) are mu-selective opiates which have been isolated from brain. M6S is an effective analgesic, with a 30-fold greater potency than morphine in the mouse radiant heat tailflick assay and similar to the active morphine metabolite morphine-6beta-glucuronide (M6G). M6S analgesia is reversed by 3-methoxynaltrexone at low antagonist doses which are inactive against morphine, suggesting that M6S may be acting through the same mechanisms as M6G. Consistent with this possibility, antisense mapping of the MOR-1 clone revealed that M6S analgesia was lowered by probes targeting exon 2 and not by targeting exon 1, a sensitivity profile similar to that of M6G and not morphine. C6S also has analgesic activity at doses approximately 10-fold greater than M6S. However, its characterization was impeded by the appearance of seizures at doses below full analgesic activity. Thus, M6S is a potent analgesic with pharmacological properties similar to M6G. C6S has limited utility due to its high level of toxicity.     

Effects of morphine and morphine withdrawal on adrenergic neurons of the rat rostral ventrolateral medulla

In urethane anesthetized rats, iontophoretic application of morphine or α-methylnoradrenaline (α-MNE) inhibited (80–100%) the discharges of all putative adrenergic (C1) cells of the rostral ventrolateral medulla (RVLM). The effect of morphine was blocked selectively by naloxone while that of α-MNE was blocked selectively by theα₂-adrenergic antagonist idazoxan. Putative C1 cells were inhibited (75–100%) by low i.v. doses of clonidine (10–15 μg/kg). Most cells (7/10) were also inhibited by morphine i.v. (81% at 7 mg/kg). Two cells were slightly excited at doses below 2 mg/kg and inhibited at higher doses. Three cells were excited only. All effects of morphine i.v. were reversed by naloxone (1 mg/kg, i.v.). Intravenous administration of naloxone to morphine-dependent rats increased significantly the firing rate of all putative C1 adrenergic cells (from 5.8 ± 0.9 spikes/s to 12.3 ± 1.5 spikes/s;n = 8). During withdrawal these cells could still be inhibited (80–100%) by i.v. injection of clonidine (15 μg/kg). C-Fos expression induced by naltrexone-precipitated withdrawal was examined in the brainstem of freely moving morphine-dependent rats pretreated with clonidine or saline before injection of the opioid antagonist. The locus coeruleus (LC) of the same rats was examined for comparison. Morphine withdrawal without clonidine treatment significantly increased the number of Fos-like-immunoreactive (Fos-LIR) cells in the RVLM and LC. Clonidine pretreatment (1 mg/kg, i.p.) reduced the number of withdrawal-activated Fos-LIR cells in LC by 81%. In the RVLM this reduction averaged 37% for all cell types and 48% for C1 adrenregic cells. Further, a very large proportion of RVLM neurons that expressed c-Fos during morphine withdrawal (83%) were immunoreactive forα₂A-adrenergic receptors. This study suggests that, like noradrenergic cells of the LC, C1 adrenergic neurons of the RVLM are: (i) inhibited by both opiate andα₂-adrenergic receptor agonists; and (ii) activated during naloxone-precipitated morphine withdrawal, Since C1 cells are considered essential to sympathetic tone generation, their inhibition by morphine may contribute to the hypotensive effects of this opioid agonist in non-dependent individuals. Their excitation during opiate withdrawal may also contribute to the autonomic activation that characterizes this syndrome. Finally, inhibition of C1 cells by clonidine may contribute to the clinically recognized efficacy of this drug to attenuate autonomic signs of opiate withdrawal.     

Cardiovascular effects elicited by central administration of physostigmine via M2 muscarinic receptors in conscious cats

The cardiovascular effects of an intracerebroventricular (i.c.v.) injection of physostigmine were studied using conscious cats. Physostigmine (5–25 μg: 5 μl) caused a dose-dependent increase in mean arterial pressure (MAP) and heart rate (HR). The highest dose (25 μg) increased MAP and HR by 32 ± 3 mmHg and 45 ± 5 beats/min, respectively (n = 5). Pre-administration of the muscarinic receptor antagonist, atropine (25 μg; i.c.v.) blocked the effects of physostigmine (25 μg; i.c.v.). Also, the pre-administration of the M₂ muscarinic antagonist, methoctramine (25 μg; i.c.v.), antagonized the cardiovascular effects of physostigmine without altering the baseline variables. However, the M₁ muscarinic antagonist, pirenzepine (100 μg; i.c.v.) did not alter baseline MAP or HR, and also failed to inhibit the cardiovascular responses to physostigmine. Similarly, the M₃ muscarinic blocker, 4-diphenyl-acetoxy-N-methylpiperidine methiodide (50 μg; i.c.v.), neither changed baseline cardiovascular variables nor blocked the effects of physostigmine. When the same cats were anesthetized with intravenous injection of sodium pentobarbital (25–30 mg/kg), physostigmine (25 μg; i.c.v.) evoked a decrease in MAP and HR of 13 ± 6 mmHg and 15 ± 6 bpm, respectively (n = 5). These results demonstrate that the increases in MAP and HR to the i.c.v. administration of physostigmine in conscious cats arepossibly mediated through stimulation of central M₂ muscarinic receptors. In addition, anesthesia reverses the effects elicited by the central administration of physostigmine to a decrease in MAP and HR.     

Morphine dependence in human neuroblastoma SH-SY5Y cells is associated with adaptive changes in both the quantity and functional interaction of PGE1 receptors and stimulatory G proteins

Chronic exposure of all-trans-retinoic acid-differentiated SH-SY5Y cells to morphine (10 μM; 2 days) results in sensitization of adenylate cyclase as characterized by a significant increase in both PGE1 receptor-mediated as well as receptor-independent (NaF, 10 mM; forskolin, 100 μM) stimulation of effector activity. To investigate the underlying biochemical alterations, chronic opioid regulation of each of the components comprising the stimulatory PGE, receptor system was examined. On receptor level, chronic morphine treatment was found to reduce PGE₁ receptor number (B_max) by approximately 40%, whereas their affinity slightly increased. Binding experiments performed in the presence of GTPγS (100 μM) further indicate that the decrease in PGE1 receptor density is associated with a loss of functionally G protein-coupled receptors. On post-receptor level, chronic morphine treatment substantially increased the abundance and functional activity of stimulatory G proteins, as assessed by cholera toxin-catalyzed ADP-ribosylation of G_sα and S49 cyc⁻ reconstitution assays. No changes were found on the level of adenylate cyclase. Evaluation of the functional interaction between PGE₁ receptors and G_s in situ by application of a C-terminal anti-G_sα antibody revealed a more intense coupling efficiency between these two entities, since a significant higher amount of antibody (2.3-fold) was required in morphine dependent cell membranes to half-maximally attenuate PGE₁ receptor-stimulated adenylate cyclase activity. In addition, limitation of the amount of functionally available G_sα within the PGE₁ receptor/adenylate cyclase signal transduction cascade abolished the generation of a supersensitive adenylate cyclase response during the state of naloxone (100 μM)-precipitated withdrawal. These data demonstrate that in human neuroblastoma SH-SY5Y cells chronic morphine-induced sensitization of adenylate cyclase is associated with distinct quantitative and qualitative adaptations within the stimulatory adenylate cyclase-coupled PGE₁ receptor system. Thus, alterations in the functional activity of stimulatory receptor systems are suggested to contribute to the cellular mechanisms underlying opioid dependence.     

Inhibition of morphine tolerance and dependence by diazepam and its relation to μ-opioid receptors in the rat brain and spinal cord

We have recently observed that concomitant administration of diazepam to morphine pellet implanted rats results in the inhibition of the development of morphine tolerance and dependence. We have now analyzed μ-opioid receptors in rats treated with morphine and diazepam for 5 days by using -DAMGO for binding studies. Male Sprague–Dawley rats were made tolerant and dependent by subcutaneous (s.c.) implantation of six morphine pellets (two pellets on the first day, and four on the second day). Diazepam (0.25 mg/kg b.wt) was injected once daily intraperitoneally (i.p.) for 5 days. Control rats were implanted with placebo pellets and injected once daily with saline or diazepam (i.p.). Animals were administered s.c. naloxone (10 mg/kg) to induce naloxone-precipitated withdrawal syndrome on the final day of the experiment (day 5). There was an up-regulation of μ-receptor (B_max increased) in the spinal cord of morphine tolerant (+139%) and dependent (+155%) rats compared to saline treated animals. Diazepam treatment abolished the up-regulation of μ-receptors in spinal cord of morphine treated rats. In the cortex, B_max was not affected in morphine tolerant or dependent rats but it decreased by 38% in morphine tolerant and 65% in morphine dependent rats treated with diazepam. The K_d of μ-receptors increased in the cortex, striatum and hypothalamus of morphine dependent rats. Diazepam treatment decreased the K_d of μ-receptors in the cortex of morphine tolerant and hypothalamus of morphine-dependent rats. These results suggest that diazepam treatment antagonizes the up-regulation of CNS μ-receptors observed in morphine tolerant rats. In addition, morphine tolerance and dependence may be associated with conversion of μ-opioid receptors to μ*-constitutive opioid receptors that are less active, and this conversion is prevented in the brain of animals treated with diazepam.     

Effects of intracerebroventricularly administered chimeric peptide of metenkephalin and FMRFa—[D-Ala]YFa—on antinociception and its modulation in mice

An enzymatically stable analog of YGGFMKKKFMRFamide (YFa), a chimeric peptide of metenkephalin and FMRFa, was synthesised. The antinociceptive effects of intracerebroventricular injections of this analog—[D-Ala²]YAGFMKKKFMRFamide ([D-Ala²]YFa)—was then investigated using the mouse radiant-heat tail-flick test. [D-Ala²]YFa produced modest to good antinociception at 1, 2, and 5 μg/mouse (0.64, 1.28, and 3.22 nmol, respectively). This antinociceptive effect was completely reversed by the opioid receptor antagonist naloxone (1.5 μg/mouse: 4.12 nmol, intracerebroventricular [i.c.v.]), administered 5 min prior. Pretreatment (5 min) with either neuropeptides FF (1 μg/mouse: 0.92 nmol, i.c.v.) or FMRFa (1 μg/mouse: 1.69 nmol, i.c.v.) significantly attenuated the antinociceptive effects induced by [D-Ala²]YFa (1 μg/mouse, i.c.v.). Intracerebroventricular administration of [D-Ala²]YFa at 1 μg/mouse dose with morphine (2 μg/mouse: 5.86 nmol, i.c.v.) produced an additive antinociceptive effect, suggesting that [D-Ala²]YFa may have a modulatory effect on opioid (morphine) analgesia. These results provide further support for a role of such amphiactive sequences in antinociception and its modulation.     

Morphine blocks the bradycardia associated with severe hemorrhage in the anesthetized rat

Progressive hemorrhage in the absence of tissue injury produces a biphasic response: an initial tachycardia, vasoconstriction and maintenance of arterial blood pressure by the baroreflex, followed by bradycardia, vasodilatation and hypotension due to the activation of a second ‘depressor’ reflex. The present study has investigated the effect of morphine (a μ-opioid receptor agonist) on the cardiac chronotropic response to a progressive hemorrhage at 2% total estimated blood volume (BV) min⁻¹ in the anesthetized rat. In control rats (20 μl saline intracerebroventricularly, i.c.v.) heart period initially decreased significantly (P < 0.05) by a maximum of 5.4 ± 0.8 ms from a baseline of 147.3 ± 2.2 ms after a blood loss of 8.3 ± 1.5% BV, and then increased significantly by a maximum of 43.0 ± 5.5 ms above the baseline after the loss of 34.5 ± 1.6% BV. Blood pressure was initially maintained and then fell during the hemorrhage. The increase in heart period was prevented by treatment with morphine (10 μg i.c.v.), and the fall in blood pressure delayed significantly. These effects of morphine were prevented by pretreatment with naloxone (20 μg i.c.v.). Intravenous (i.v.) administration of morphine (10 μg) had no effect on the response to hemorrhage. However, a clinically relevant dose of 0.5 mg · kg⁻¹ morphine (i.v.) abolished the bradycardia and delayed the fall in blood pressure associated with hemorrhage. These results indicate that morphine, acting on central nervous opioid receptors, can abolish the bradycardia and delay the hypotension associated with progressive hemorrhage, a pattern of response reminiscent of the effects of musculo-skeletal injury on the response to blood loss.     

Medullary μ and δ opioid receptors modulate mesencephalic morphine analgesia in rats

Supraspinal opioid analgesia is mediated in part by connections between the midbrain periaqueductal gray (PAG) and rostral ventral medulla (RVM) which includes the nuclei raphe magnus and reticularis gigantocellularis. Serotonergic 5HT₂ and 5HT₃ receptor subtypes appear to participate in this pathway since general and selective serotonergic antagonists microinjected into the RVM significantly reduced morphine analgesia elicited from the PAG. Since both an enkephalinergic pathway between the PAG and RVM and intrinsic enkephalinergic cells in the RVM exist, the present study evaluated the abilities of general (naltrexone), μ-selective (β-funaltrexamine: B-FNA) andδ₂-selective (naltrindole) opioid receptor subtype antagonists microinjected into the RVM to alter morphine (2.5 μg) analgesia elicited from the PAG as measured by the tail-flick and jump tests. Mesencephalic morphine analgesia was significantly reduced after pretreatment in the RVM with naltrexone (1–10 μg), B-FNA (0.5–5 μg) or naltrindole (0.5–5 μg). Naltrexone in the RVM failed to alter basal nociceptive thresholds and none of the opioid antagonists were effective in reducing mesencephalic morphine analgesia when they were microinjected into placements lateral or dorsal to the RVM. These data indicate that μ andδ₂ opioid receptors in the RVM modulate the transmission of opioid pain-inhibitory signals from the PAG.     

Involvement of glial cell line-derived neurotrophic factor in inhibitory effects of a hydrophobic dipeptide Leu-Ile on morphine-induced sensitization and rewarding effects

There are few efficacious medications for drug dependence at present. We have previously demonstrated that Leu-Ile, which induces the expression of not only tumor necrosis factor-alpha (TNF-alpha) but also glial cell line-derived neurotrophic factor (GDNF), inhibits methamphetamine (METH) and morphine (MOR)-induced sensitization and rewarding effects by regulating extracellular dopamine levels via the induction of TNF-alpha expression, and indicated the potential of Leu-Ile as a novel therapeutic agent for METH and MOR-induced dependence. In the present study, we investigated the involvement of GDNF in inhibitory effects of Leu-Ile on MOR-induced sensitization and rewarding effects. Repeated treatment with MOR for 9 days, which results in an enhancement of the locomotor-stimulating effects (sensitization) of MOR, increased GDNF levels in the nucleus accumbens compared with those in saline-treated mice. Repeated pre-treatment with Leu-Ile for 9 days potentiated the MOR-induced increase in GDNF levels. MOR at a low dose (3mg/kg) produced place preference in GDNF heterozygous knockout (GDNF-(+/-)) mice, but not in littermate GDNF-(+/+) mice. No inhibitory effect of Leu-Ile on MOR-induced place preference was observed in GDNF-(+/-) mice. These results suggest that GDNF is involved in the inhibitory effects of Leu-Ile on MOR-induced sensitization and rewarding effects.     

Role of central opioid receptor subtypes in morphine-induced alterations in peripheral lymphocyte activity

Morphine has been shown to decrease proliferative responses of rat T-lymphocytes via central opioid receptors, however, the specific receptor subtype(s) mediating this effect have not been established. To determine the potential role of central μ opioid receptors in morphine-mediated suppression of T-lymphocyte proliferation, 20 nmol/2 μl of either morphine sulfate or DAMGO (μ-selective agonist) were administered into the lateral ventricle of freely moving Sprague–Dawley rats. Lymphocyte proliferative response to the polyclonal T cell mitogen concanavalin A (ConA), changes in splenic natural killer cell (NK) cytolytic activity, activation of the hypothalamic–pituitary–adrenal (HPA) axis and antinociception (tail-flick latency) were examined. Results indicated that like morphine, DAMGO decreased blood lymphocyte proliferative responses by 80% and elevated both tail-flick latency and plasma corticosterone when compared to saline-treated animals. The proliferation response of lymphocytes from the spleen or thymus and splenic NK cell activity were not significantly altered by either morphine or DAMGO treatment. The effects of DAMGO were determined to be dose-dependent and completely antagonized by naltrexone pretreatment. Central administration of DPDPE (δ-selective agonist) and U-50488 (κ-selective agonist) produced between 40–50% suppression of blood lymphocyte proliferation responses only at a dose five times greater (100 nmol) than DAMGO treatment, without altering antinociception or activation of the HPA axis. To determine the central opioid receptor subtype(s) involved in the effects of morphine, selective opioid antagonists were microinjected into the lateral ventricle prior to morphine treatment (6 mg/kg, s.c.). CTOP (μ-selective antagonist, 5 μg/2 μl) completely blocked the effects of morphine on all parameters measured, however, naltrindole (δ-selective antagonist, 2 μg/2 μl) or nor-binaltorphimine (κ-selective antagonist, 73.5 μg/2 μl) failed to block the effects of morphine. Collectively, these results provide evidence that morphine acts primarily through central μ receptors to modulate peripheral blood lymphocyte proliferation responses. Further, the antinociception and blood lymphocyte effects show greater sensitivity to opioids than either natural killer cell cytolytic activity or activation of the HPA axis.     

Morphine and morphine-6beta-glucuronide-induced feeding are differentially reduced by G-protein alpha-subunit antisense probes in rats

Although morphine and its active metabolite, morphine-6beta-glucuronide (M6G), each induce mu-opioid receptor-sensitive feeding, different antisense oligodeoxynucleotide (AS ODN) probes directed against the MOR-1 clone produce distinct effects. Thus, MOR-1 AS ODN probes directed against exons 1 or 4 reduce morphine-, but not M6G-induced feeding, whereas probes directed against exons 2 or 3 reduce M6G-, but not morphine-induced feeding. AS ODN probes directed against different G-protein alpha-subunits differentially reduced morphine (G(ialpha2)) and M6G (G(ialpha1))-induced analgesia. The present study evaluated the ability of AS ODN probes directed against G-protein alpha-subunits to reduce feeding induced by morphine and M6G in rats. The AS ODN probes (25 microg, i.c.v.) were administered once 24 h prior to morphine (5 microg, i.c.v.) or M6G (250 ng) and spontaneous free feeding was assessed 1, 2 and 4 h thereafter. In agreement with analgesic studies, morphine-induced feeding was significantly reduced by the G(ialpha2) AS ODN probe. Morphine-induced feeding was unaffected by AS ODN probes directed against either G(ialpha1), G(ialpha3), G(oalpha), G(x/zalpha), G(qalpha) or a nonsense control probe, and was significantly enhanced by pretreatment with the G(salpha) probe. In contrast, M6G-induced feeding was significantly reduced by AS ODN probes directed against either G(ialpha1), G(ialpha3) or G(x/zalpha), whereas AS ODN probes targeting G(ialpha2), G(oalpha), G(salpha), G(qalpha) or a nonsense control probe were ineffective. When M6G-induced feeding was assessed at a dose (500 ng) which was sensitive to MOR-1 AS ODN effects, none of the G-protein alpha-subunit AS ODN probes were effective. These data indicate that morphine and M6G-induced feeding are mediated through different G-protein alpha-subunits, and provide further evidence for separate and distinct molecular mechanisms mediating these functional responses through different opioid receptors. This strongly suggests that M6G may act through a novel opioid receptor displaying a distinct pharmacological mechanism.