Microinjection of 26RFa, an endogenous ligand for the glutamine RF-amide peptide receptor (QRFP receptor), into the rostral ventromedial medulla (RVM), locus coelureus (LC), and periaqueductal grey (PAG) produces an analgesic effect in rats.

26RFa is an endogenous ligand for the QRFP receptor. We previously found that intracerebroventricular injection of 26RFa produces an analgesic effect in a rat formalin test. In the present study, we directly tested the hypothesis that the analgesic effects of 26RFa in the formalin test are mediated in well-recognized regions of the descending inhibitory pain pathways, such as the rostral ventromedial medulla (RVM), locus coeruleus (LC), and periaqueductal grey (PAG) in rats. Injection cannulae were stereotaxically placed in the RVM, LC, or PAG through a burr hole. 26RFa (15 µg) or saline was delivered in a total volume of 0.5 µL. In a formalin test, 50 µL of 5% formalin was injected subcutaneously into the hind paw. In an antagonist study, idazoxan, an α-2 antagonist, or naloxone, an opioid receptor antagonist, was administered. Microinjection of 26RFa into the RVM had no effect compared with that in saline-injected rats. Microinjection of 26RFa into the LC contralateral, but not ipsilateral, to the formalin injection site significantly decreased the number of flinching behaviors compared with that of saline-injected rats. This effect was antagonized by intrathecal injection of idazoxan. Microinjection of 26RFa into the contralateral, but not ipsilateral, PAG produced an analgesic effect, and this effect was partly antagonized by intraperitoneal naloxone. These data suggest that 26RFa microinjected into the contralateral LC induced noradrenaline release in the spinal cord and produced an analgesic effect. In the contralateral PAG, 26RFa activated the opioid system, and some analgesic effects were mediated by opioid system activation.

In vivo and in vitro attenuation of naloxone-precipitated experimental opioid withdrawal syndrome by insulin and selective KATP channel modulator.

RATIONALE: Opiate exposure for longer duration develops state of dependence in humans and animals, which is revealed by signs and symptoms of withdrawal precipitated by opioid receptor antagonists. The sudden withdrawal of opioids produces a withdrawal syndrome in opioid-dependent subjects. Insulin and ATP-sensitive potassium (KATP) channel-mediated glucose homeostasis have been shown to modulate morphine withdrawal. OBJECTIVE: Present study has been structured to investigate the role of insulin and pharmacological modulator of KATP channel (gliclazide) in experimental morphine withdrawal syndrome, both invivo and invitro. METHODS: In this study, naloxone-precipitated morphine withdrawal syndrome in mice (invivo) as well as in rat ileum (invitro) were utilized to assess opioid withdrawal phenomenon. Morphine withdrawal syndromes like jumping and rearing frequency, forepaw licking, circling, fore paw tremor, wet dog shake, sneezing, overall morphine withdrawal severity (OMWS), serum glucose, brain malondialdehyde (MDA), glutathione (GSH), nitrite/nitrate, and calcium (Ca(+2)) were assessed. RESULTS: Naloxone has significantly increased morphine withdrawal syndrome, both invivo and invitro. Insulin and gliclazide have significantly attenuated, naloxone induced behavioral changes like jumping and rearing frequency, forepaw licking, wet dog shake, sneezing, straightening, circling, OMWS, and various biochemical impairments such as serum glucose, brain MDA, GSH, nitrite/nitrate, and Ca(+2) in morphine-dependent animals (invivo). In vitro, insulin and gliclazide have significantly reduced naloxone-induced contraction in morphine-withdrawn rat ileum preparation. CONCLUSIONS: Insulin and gliclazide (KATP channel blocker) have attenuated naloxone-precipitated morphine withdrawal syndrome, both invivo and invitro. Thus, insulin and KATP channel modulation may provide new avenues for research in morphine withdrawal.

Effect of naloxone on ischemic acute kidney injury in the mouse.

Renal ischemia produces sympathoexcitation, which is responsible for the development of ischemic acute kidney injury. Stimulation of central opioid receptors activates the renal sympathetic nerve. The present study examined the effect of an opioid receptor antagonist naloxone on the ischemia/reperfusion-induced renal dysfunction in mice. Blood urea nitrogen (BUN) and plasma creatinine increased 24 h after the renal ischemia/reperfusion. Intraperitoneal or intracerebroventricular, but not intrathecal, pretreatment with naloxone suppressed the renal ischemia/reperfusion-induced increases in BUN and plasma creatinine. This effect of naloxone was reversed by subcutaneous pretreatment with morphine. Selective MOP receptor antagonist ß-funaltrexamine (FNA) also suppressed the renal ischemia/reperfusion-induced increases in BUN and plasma creatinine. Moreover, tyrosine hydroxylase expression in the renal tissue increased 24 h after renal ischemia/reperfusion, which was abolished by intraperitoneal or intracerebroventricular pretreatment with naloxone and FNA. Immunohistochemical experiments revealed a significant increase in the number of the Fos family proteins (c-Fos, FosB, Fra-1, and Fra-2) positive cells in the paraventricular nucleus of hypothalamus and supraoptic nucleus 24 h after the renal ischemia/reperfusion. Intracerebroventricular pretreatment with naloxone attenuated the renal ischemia/reperfusion-induced increase in the number of the Fos family proteins positive cells in these areas. Finally, we observed that i.c.v. pretreatment with antiserum against ß-endorphin also suppressed the increased blood urea and plasma creatinine. These results suggest that the blockade of central opioid receptors can attenuate the ischemic acute kidney injury through the inhibition of renal sympathoexcitation. The central opioid receptors may thus be a new target for the treatment of ischemic organ failures.

Phenylmethanesulfonyl fluoride, a serine protease inhibitor, suppresses naloxone-precipitated withdrawal jumping in morphine-dependent mice.

We have previously shown that intracerebroventricular (i.c.v.) administration of cysteine protease inhibitors suppresses naloxone-precipitated withdrawal jumping in morphine-dependent mice, presumably through the inhibition of dynorphin degradation (see (Tan-No, K., Sato, T., Shimoda, M., Nakagawasai, O., Niijima, F., Kawamura, S., Furuta, S., Sato, T., Satoh, S., Silberring, J., Terenius, L., Tadano, T., 2010. Suppressive effects by cysteine protease inhibitors on naloxone-precipitated withdrawal jumping in morphine-dependent mice. Neuropeptides 44, 279-283)). In the present study, we examined the effect of phenylmethanesulfonyl fluoride (PMSF), a serine protease inhibitor, on naloxone-precipitated withdrawal jumping in morphine-dependent mice. The doses of morphine (mg/kg per injection) were subcutaneously given twice daily for 2 days [day 1 (30) and day 2 (60)]. On day 3, naloxone (8 mg/kg) was intraperitoneally administered 3h after the final injection of morphine (60 mg/kg), and the number of jumps was immediately recorded for 20 min. Naloxone-precipitated withdrawal jumping was significantly suppressed by i.c.v. administration of PMSF (4 nmol), given 5 min before each morphine treatment during the induction phase, with none given on the test day. The expression of tissue plasminogen activator (tPA), a serine protease that converts plasminogen to plasmin, in the prefrontal cortex was significantly increased in morphine-dependent and -withdrawal mice, as compared with saline-treated mice. Moreover, trans-4-(aminomethyl)-cyclohexanecarboxylic acid (300 pmol), an antiplasmin agent, and (Tyr(1))-thrombin receptor activating peptide 7 (0.45 and 2 nmol), an antagonist of protease activated receptor-1 (PAR-1), significantly suppressed naloxone-precipitated withdrawal jumping. The present results suggest that PMSF suppresses naloxone-precipitated withdrawal jumping in morphine-dependent mice, presumably through the inhibition of activities of tPA and plasmin belonging to the serine proteases family, which subsequently activates PAR-1.

Opioid receptor heteromers in analgesia.

Opiates such as morphine and fentanyl, a major class of analgesics used in the clinical management of pain, exert their effects through the activation of opioid receptors. Opioids are among the most commonly prescribed and frequently abused drugs in the USA; however, the prolonged use of opiates often leads to the development of tolerance and addiction. Although blockade of opioid receptors with antagonists such as naltrexone and naloxone can lessen addictive impulses and facilitate recovery from overdose, systemic disruption of endogenous opioid receptor signalling through the use of these antagonistic drugs can have severe side effects. In the light of these challenges, current efforts have focused on identifying new therapeutic targets that selectively and specifically modulate opioid receptor signalling and function so as to achieve analgesia without the adverse effects associated with chronic opiate use. We have previously reported that opioid receptors interact with each other to form heteromeric complexes and that these interactions affect morphine signalling. Since chronic morphine administration leads to an enhanced level of these heteromers, these opioid receptor heteromeric complexes represent novel therapeutic targets for the treatment of pain and opiate addiction. In this review, we discuss the role of heteromeric opioid receptor complexes with a focus on mu opioid receptor (MOR) and delta opioid receptor (DOR) heteromers. We also highlight the evidence for altered pharmacological properties of opioid ligands and changes in ligand function resulting from the heteromer formation.

Ammonium pyrrolidine dithiocarbamate and RS 102895 attenuate opioid withdrawal in vivo and in vitro.

RATIONALE: Recently, nuclear factor kappa B is indicated in the precipitation of opioid withdrawal syndrome. NF-κB activation is noted to control the transcription and biochemical activation of chemokines. Opioid receptor activation-linked chemokine stimulation is reported to mediate certain effects produced by prolonged opioid treatment. Ammonium pyrrolidine dithiocarbamate (APD) and RS 102895 are relatively selective inhibitors of NF-κB and C-C chemokine receptor 2, respectively. OBJECTIVES: The present study investigates the effect of APD and RS 102895 on morphine withdrawal signs in vitro and in vivo. MATERIALS AND METHODS: Morphine was administered twice daily for 5 days, following which a single day 6 injection of naloxone (8 mg/kg, i.p.) precipitated opioid withdrawal syndrome in mice. Withdrawal syndrome was quantitatively assessed in terms of withdrawal severity score and the frequency of jumping, rearing, fore paw licking and circling. Naloxone-induced contraction in morphine-withdrawn isolated rat ileum was employed as an in vitro model. An isobolographic study design was employed in the two models to assess potential synergistic activity between APD and RS 102895. RESULTS: APD and RS 102895 dose-dependently attenuated naloxone-induced morphine withdrawal syndrome both in vivo and in vitro. APD was also observed to exert a synergistic interaction with RS 102895. CONCLUSIONS: It is concluded that APD and RS 102895 attenuate morphine withdrawal signs possibly by a NF-κB and C-C chemokine receptor 2 activation pathway-linked mechanisms potentially in an interdependent manner.

Effects and mechanisms of electroacupuncture on glucagon-induced small intestinal hypomotility in dogs.

BACKGROUND: Little is known on the effect of electroacupuncture (EA) (Br Med J, 2, 1976, 1225) on intestinal motility. The aim of this study was to investigate effects and mechanisms of EA on small intestinal contractions, transit, and slow waves in dogs. METHODS: Six dogs were equipped with two intestinal cannulas for the measurement of small intestinal contractions and transit. Glucagon was used to induce postprandial intestinal hypomotility. Each dog was studied in five randomized sessions: Control, glucagon, glucagon + EA, glucagon + EA + naloxone, and glucagon + EA + atropine. KEY RESULTS: 1 In the fasting state, EA induced intestinal contractions during motor quiescence (contractile index or CI: 4.4 ± 0.8 VS 8.3 ± 0.7, P < 0.05). 2 In the fed state, EA improved glucagon-induced intestinal hypomotility (CI: 3.8 ± 0.4 VS 6.1 ± 0.6, P < 0.05). 3 Electroacupuncture accelerated intestinal transit delayed by glucagon (67.9 ± 4.3 VS 40.2 ± 5.0 min, P < 0.05). 4 There was a negative correlation between the CI and the total transit time (R(2) = 0.59, P < 0.05). 5 The excitatory effect of EA was blocked by naloxone and partially blocked by atropine. 6 The percentage of normal slow waves was reduced with glucagon (70 ± 2%VS 98 ± 1% at baseline, P = 0.0015). Electroacupuncture normalized impaired slow waves and the effect was blocked by naloxone. CONCLUSIONS & INFERENCES: Electroacupuncture enhances intestinal contractions during Phase I of the migrating motor complex and glucagon-induced hypomotility in the fed state, and accelerates intestinal transit via the opioid and cholinergic pathways in dogs. Electroacupuncture may have a therapeutic potential for intestinal hypomotility.

Acylated ghrelin inhibits spontaneous luteinizing hormone pulsatility and responsiveness to naloxone but not that to gonadotropin-releasing hormone in young men: evidence for a central inhibitory action of ghrelin on the gonadal axis.

CONTEXT: Recent evidence suggests that ghrelin exerts a negative modulation on the gonadal axis. Ghrelin was reported to suppress LH secretion in both animal and human models. Moreover, acylated ghrelin (AG) also decreases the LH responsiveness to GnRH in vitro. OBJECTIVE: The objective of the study was to evaluate the effects of AG infusion on spontaneous and stimulated gonadotropin secretion. DESIGN, PARTICIPANTS, AND INTERVENTION: In seven young healthy male volunteers (age mean +/- sem 26.4 +/- 2.6 yr), we evaluated LH and FSH levels every 15 min during: 1) iv isotonic saline infusion; 2) iv saline followed by AG; LH and FSH response to GnRH (100 microg iv as a bolus), 3) alone and 4) during AG infusion; LH and FSH response to naloxone (0.1 mg/kg iv as a slow bolus), 5) alone and 6) during AG infusion. RESULTS: Significant LH but not FSH pulses were recorded in all subjects under saline infusion. AG infusion inhibited LH levels [area under the curve((240-480)): 415.8 +/- 69.7 mIU/ml.min during AG vs. 744.6 +/- 120.0 mIU/ml.min during saline, P < 0.02] and abolished LH pulsatility. No change in FSH secretion was recorded. The LH and FSH responses to GnRH during saline were not affected by AG administration. However, AG inhibited the LH response to naloxone [area under the curve ((120-210)): 229.9 +/- 39.3 mIU/ml.min during AG vs. 401.1 +/- 44.6 mIU/ml.min during saline, P < 0.01]. FSH levels were not modified by naloxone alone or in combination with AG. CONCLUSIONS: AG inhibits both spontaneous LH pulsatility and the LH response to naloxone. Because AG does not affect the LH response to GnRH, these findings indicate that the ghrelin system mediates central inhibition of the gonadal axis.

[Comparative analysis of analgesic activities of dermorphin, [DPro6]-dermorphin, and their C-terminal tripeptides].

Analgesic activities of dermorphin (DM), [DPro6]-DM, and their C-terminal tripeptides were comparatively studied. Analgesic activity was evaluated in tail flick, hot plate, tail pinch, formalin, and acetic acid writhing tests describing different levels of organization of pain sensitivity. Intraperitoneal administration of the peptides decreased the pain threshold in all these tests. The C-terminal tripeptides DM(5-7) and [DPro6]-DM(5-7) demonstrated analgesics activity comparable or sometimes higher than that of the full-length molecules. The effect of DM, [DPro6]-DM, and C-terminals fragments DM(5-7) and [DPro6]-DM(5-7) decreased after co-administration with naloxone, which points to the opioid nature of analgesic activity of the peptides.

A long-form alpha-neurotoxin from cobra venom produces potent opioid-independent analgesia.

AIM: In light of the antinociceptive activity of the short-chain neurotoxin, cobrotoxin, and other acetylcholine antagonists, the antinociceptive activity and mechanisms of cobratoxin (CTX), a long-chain postsynaptic alpha-neurotoxin, was investigated in rodent pain models. METHODS: CTX was administered intraperitoneally (30, 45, 68 microg/kg), intra-cerebral ventricularly (4.5 microg/kg) or microinjected into periaqueductal gray (PAG; 4.5 microg/kg). The antinociceptive action was tested using the hot-plate and acetic acid writhing tests in mice and rats. The involvement of the cholinergic system and opioid system in CTX-induced analgesia was examined by pretreatment of animals with atropine (0.5 mg/kg, im; or 10 mg/kg, ip) or naloxone (1 and 5 mg/kg, ip). The effect of CTX on motor activity was tested using the Animex test. RESULTS: CTX exhibited a dose-dependent analgesic action in mice as determined by both the hot-plate and acetic acid writhing tests. The peak effect of analgesia was seen 3 h after administration. In the mouse acetic acid writhing test, the intra-cerebral ventricular administration of CTX at 4.5 microg/kg (1/12th of a systemic dose) produced marked analgesic effects. Microinjection of CTX (4.5 microg/kg) into the PAG region did not elicit an analgesic action in rats in the hot-plate test. Atropine at 0.5 mg/kg (im) and naloxone at 1 and 5 mg/kg (ip) both failed to block the analgesic effects of CTX, but atropine at 10 mg/kg (ip) did antagonize the analgesia mediated by CTX in the mouse acetic acid writhing test. Acetylsalicylic acid (300 mg/kg) did not enhance the analgesic effects of CTX. At the highest effective dose of 68 microg/kg the neurotoxin did not change the spontaneous mobility of mice. CONCLUSION: CTX has analgesic effects, which are mediated in the central nervous system though not through the PAG. The central cholinergic system but not opioid system appears to be involved in the antinociceptive action of CTX.

A role for endocannabinoids in viral-induced dyskinetic and convulsive phenomena.

Dyskinesias and seizures are both medically refractory disorders for which cannabinoid-based treatments have shown early promise as primary or adjunctive therapy. Using the Borna disease (BD) virus rat, an animal model of viral encephalopathy with spontaneous hyperkinetic movements and seizure susceptibility, we identified a key role for endocannabinoids in the maintenance of a balanced tone of activity in extrapyramidal and limbic circuits. BD rats showed significant elevations of the endocannabinoid anandamide in subthalamic nucleus, a relay nucleus compromised in hyperkinetic disorders. While direct and indirect cannabinoid agonists had limited motor effects in BD rats, abrupt reductions of endocannabinoid tone by the CB1 antagonist SR141716A (0.3 mg/kg, i.p.) caused seizures characterized by myoclonic jerks time-locked to periodic spike/sharp wave discharges on hippocampal electroencephalography. The general opiate antagonist naloxone (NLX) (1 mg/kg, s.c.), another pharmacologic treatment with potential efficacy in dyskinesias or L-DOPA motor complications, produced similar seizures. No changes in anandamide levels in hippocampus and amygdala were found in convulsing NLX-treated BD rats. In contrast, NLX significantly increased anandamide levels in the same areas of normal uninfected animals, possibly protecting against seizures. Pretreatment with the anandamide transport blocker AM404 (20 mg/kg, i.p.) prevented NLX-induced seizures. These findings are consistent with an anticonvulsant role for endocannabinoids, counteracting aberrant firing produced by convulsive agents, and with a functional or reciprocal relation between opioid and cannabinoid tone with respect to limbic convulsive phenomena.

Differentiation of opioid receptor preference by [Dmt1]endomorphin-2-mediated antinociception in the mouse.

The potent opioid [Dmt1]endomorphin-2 (Dmt-Pro-Phe-Phe-NH2) differentiated between the opioid receptor subtypes responsible for the antinociception elicited by endomorphin-2 in mice. Antinociception, induced by the intracerebroventricular administration of [Dmt1]endomorphin-2 and inhibited by various opioid receptor antagonists [naloxone, naltrindole, beta-funaltrexamine, naloxonazine], was determined by the tail-flick (spinal effect) and hot-plate (supraspinal effect) tests. The opioid receptor subtypes involved in [Dmt1]endomorphin-2-induced antinociception differed between these in vivo model paradigms: naloxone (non-specific opioid receptor antagonist) and beta-funaltrexamine (irreversible mu1/mu2-opioid receptor antagonist) blocked antinociception in both tests, although stronger inhibition occurred in the hot-plate than the tail-flick test suggesting involvement of other opioid receptors. Consequently, we applied naloxonazine (mu1-opioid receptor antagonist) that significantly blocked the effect in the hot-plate test and naltrindole (delta-opioid receptor antagonist), which was only effective in the tail-flick test. The data indicated that [Dmt1]endomorphin-2-induced spinal antinociception was primarily mediated by both mu2- and delta-opioid receptors, while a supraspinal mechanism involved only mu1/mu2-subtypes.

The terrestrial Gastropoda Megalobulimus abbreviatus as a useful model for nociceptive experiments: effects of morphine and naloxone on thermal avoidance behavior.

We describe the behavior of the snail Megalobulimus abbreviatus upon receiving thermal stimuli and the effects of pretreatment with morphine and naloxone on behavior after a thermal stimulus, in order to establish a useful model for nociceptive experiments. Snails submitted to non-functional (22 degrees C) and non-thermal hot-plate stress (30 degrees C) only displayed exploratory behavior. However, the animals submitted to a thermal stimulus (50 degrees C) displayed biphasic avoidance behavior. Latency was measured from the time the animal was placed on the hot plate to the time when the animal lifted the head-foot complex 1 cm from the substrate, indicating aversive thermal behavior. Other animals were pretreated with morphine (5, 10, 20 mg/kg) or naloxone (2.5, 5.0, 7.5 mg/kg) 15 min prior to receiving a thermal stimulus (50 degrees C; N = 9 in each group). The results (means +/- SD) showed an extremely significant difference in response latency between the group treated with 20 mg/kg morphine (63.18 +/- 14.47 s) and the other experimental groups (P < 0.001). With 2.5 mg/kg (16.26 +/- 3.19 s), 5.0 mg/kg (11.53 +/- 1.64 s) and 7.5 mg/kg naloxone (7.38 +/- 1.6 s), there was a significant, not dose-dependent decrease in latency compared to the control (33.44 +/- 8.53 s) and saline groups (29.1 +/- 9.91 s). No statistically significant difference was found between the naloxone-treated groups. With naloxone plus morphine, there was a significant decrease in latency when compared to all other groups (minimum 64% in the saline group and maximum 83.2% decrease in the morphine group). These results provide evidence of the involvement of endogenous opioid peptides in the control of thermal withdrawal behavior in this snail, and reveal a stereotyped and reproducible avoidance behavior for this snail species, which could be studied in other pharmacological and neurophysiological studies.

The terrestrial Gastropoda Megalobulimus abbreviatus as a useful model for nociceptive experiments: effects of morphine and naloxone on thermal avoidance behavior

We describe the behavior of the snail Megalobulimus abbreviatus upon receiving thermal stimuli and the effects of pretreatment with morphine and naloxone on behavior after a thermal stimulus, in order to establish a useful model for nociceptive experiments. Snails submitted to non-functional (22°C) and non-thermal hot-plate stress (30°C) only displayed exploratory behavior. However, the animals submitted to a thermal stimulus (50°C) displayed biphasic avoidance behavior. Latency was measured from the time the animal was placed on the hot plate to the time when the animal lifted the head-foot complex 1 cm from the substrate, indicating aversive thermal behavior. Other animals were pretreated with morphine (5, 10, 20 mg/kg) or naloxone (2.5, 5.0, 7.5 mg/kg) 15 min prior to receiving a thermal stimulus (50°C; N = 9 in each group). The results (means ± SD) showed an extremely significant difference in response latency between the group treated with 20 mg/kg morphine (63.18 ± 14.47 s) and the other experimental groups (P < 0.001). With 2.5 mg/kg (16.26 ± 3.19 s), 5.0 mg/kg (11.53 ± 1.64 s) and 7.5 mg/kg naloxone (7.38 ± 1.6 s), there was a significant, not dose-dependent decrease in latency compared to the control (33.44 ± 8.53 s) and saline groups (29.1 ± 9.91 s). No statistically significant difference was found between the naloxone-treated groups. With naloxone plus morphine, there was a significant decrease in latency when compared to all other groups (minimum 64 percent in the saline group and maximum 83.2 percent decrease in the morphine group). These results provide evidence of the involvement of endogenous opioid peptides in the control of thermal withdrawal behavior in this snail, and reveal a stereotyped and reproducible avoidance behavior for this snail species, which could be studied in other pharmacological and neurophysiological studies.

GHB ameliorates naloxone-induced conditioned place aversion and physical aspects of morphine withdrawal in mice.

RATIONALE: Gamma-hydroxybutyric acid (GHB) is a naturally occurring substance in the brain, the administration of which has proved useful in the treatment of the opiate withdrawal symptoms in humans. OBJECTIVES: The aim of the present work was to validate this beneficial effect on the physical and motivational aspects of morphine withdrawal in mice. METHODS: In a first experiment, animals rendered morphine-dependent were conditioned to develop a place aversion (CPA) to the compartment paired with naloxone administration in a two-chamber apparatus. The conditioning phase consisted of three pairings of either naloxone (0.250 mg/kg) or vehicle in one compartment, both with similar time allotments during the preconditioning test. During the testing phase, mice were again allowed to explore the entire apparatus. GHB (6, 12.5, 25, and 50 mg/kg) was administered during either the acquisition or expression phase of this conditioning. In a second experiment, the capacity of GHB to ameliorate the intensity of physical signs of morphine withdrawal was evaluated. RESULTS: GHB blocked CPA in both phases: administered during acquisition (from 12.5 mg/kg and higher) as well as in the expression phase (from 6 mg/kg, except for 25 mg/kg). It also decreased the intensity of physical signs of morphine withdrawal to near control levels measured by the modified Gellert-Holtzman scale (25 mg/kg and higher). Decreases in jumping, body shakes, and paw tremor were also observed. CONCLUSIONS: Our results support the idea that GHB ameliorates both aspects of morphine withdrawal, physical as well as motivational signs.

4-Caffeoyl-1,5-quinide in roasted coffee inhibits [3H]naloxone binding and reverses anti-nociceptive effects of morphine in mice.

RATIONALE: Cinnamoylquinides are formed from the corresponding chlorogenic acids during coffee roasting. Instant coffee has been shown to displace binding of the mu opioid receptor antagonist, [3H]naloxone, but the putative active agent, feruloylquinide, has not been characterized. OBJECTIVES: The goal was to identify the active agent(s) in coffee by measuring the binding affinity of individual cinnamoyl-1,5-quinides to the human mu opioid receptor, and determine the effects of these compounds on morphine-induced anti-nociceptive behavior in mice. METHODS: Cinnamoyl-1,5-quinides in extracts of decaffeinated instant coffee were quantified by reverse-phase HPLC comparisons with synthetic samples of 3-coumaroyl-1,5-quinide and 4-coumaroyl-1,5-quinide, 3-caffeoyl-1,5-quinide and 4-caffeoyl-1,5-quinide (4-CQL) 3-feruloyl-1,5-quinide and 4-feruloyl-1,5-quinides and 3,4-dicaffeoyl-1,5-quinide (DICAQ). Affinities of the cinnamoyl-1,5-quinides and decaffeinated instant coffee extract were determined by displacement of [3H]naloxone binding in cultured HEK-MOR cells. Inhibition of the anti-nociceptive activity of morphine (1 mg/kg IP) was determined in C57BL/6J mice using the hot plate test at 52 degrees C. RESULTS: Extract of decaffeinated instant coffee produced a displacement K(i) of 42+/-16 mg/l, while the K(i) of a synthetic sample of 4-CQL was 4.4+/-0.4 microM. Compounds with a cinnamoyl substituent in the 4-position of the quinide, i.e. 4-CQL, DICAQ, 3,4-diferuloyl-1,5-quinide, and 3,4-dicoumaroyl-1,5-quinide, had affinities for the mu opioid receptor in the low micromolar range. In the hot plate test, coffee extract, containing 0.78% of 4-CQL, reversed the anti-nociceptive effect of morphine at 10 mg/kg IP. Two cinnamoyl-1,5-quinides found in roasted coffee, DICAQ, and 4-CQL, were active at 1 and 0.1 mg/kg IP, respectively. CONCLUSIONS: These results suggest that the previously reported anti-opioid activity of instant coffee is caused primarily by the presence of 4-CQL, and to lesser extent by other cinnamoyl-1,5-quinides.

Heparan sulfate proteoglycans are involved in opiate receptor-mediated cell migration.

Opioid receptors are expressed in cells of the immune system, and potent immunomodulatory effects of their natural and synthetic ligands have been reported. In some studies, the opiate receptor antagonist naloxone itself displayed immunomodulatory actions. We investigated effects of naloxone on leukocyte chemotaxis. Cell migration was tested in micropore filter assays using modified Boyden chambers, and receptor expression was investigated using radiolabel binding assays. Naloxone induced peripheral blood nonadherent mononuclear cell and neutrophil chemotaxis at nanomolar concentrations and deactivated their migration toward beta-endorphin, angiotensin II, somatostatin, or interleukin-8 but not toward RANTES, vasoactive intestinal peptide, or substance P. Ligand binding studies showed no alteration in the binding of interleukin-8 to neutrophils by naloxone. Cleavage of heparan sulfate from proteoglycans on the cells' surface completely inhibited chemotactic and deactivating properties of naloxone but not other attractants. Chemotactic properties were abolished by pretreating cells with heparinase, chondroitinase, sodium chlorate, and anti-syndecan-4 antibodies, indicating the involvement of syndecan-4. The extent of migration toward naloxone was diminished by pretreatment with dimethylsphingosine, a specific sphingosine kinase inhibitor. As syndecan-4 signaling in leukocyte chemotaxis involves activation of sphingosine kinase, results indicate that naloxone interacts with syndecan-4 function in cell migration and suggest a role for heparan sulfate proteoglycans as coreceptors to members of the delta-opiate receptor family.

Nicotine attenuates place aversion induced by naloxone in single-dose, morphine-treated rats.

RATIONALE: Acute physical dependence refers to the withdrawal syndrome precipitated by an opioid antagonist administered several hours after either a single dose or a short-term infusion of an opioid agonist. OBJECTIVES: We examined the mechanism of nicotine-induced attenuation of naloxone-precipitated withdrawal syndrome when used to produce an aversive motivational state in a place-conditioning paradigm. METHODS: The effect of nicotine was investigated through place aversion induced by naloxone in morphine-pretreated rats. Additionally, the mechanism of nicotine action in this model was explored specifically in relation to the dopaminergic system through the use of dopamine receptor antagonist and agonist. RESULTS: Place avoidance behavior was potently elicited by naloxone (0.5 mg/kg s.c.) 24 h after a single exposure to morphine (10 mg/kg s.c.). Avoidance behavior was attenuated by pretreatment with a 0.2-mg/kg dose of nicotine 15 min prior to naloxone administration. The effect of nicotine was completely blocked by mecamylamine, but not hexamethonium. The dopamine receptor antagonists haloperidol (0.05, 0.1 mg/kg, s.c.), SCH23390 (0.1 mg/kg, s.c.), raclopride (1.0 mg/kg, s.c.) and eticlopride (0.1 mg/kg, s.c.) showed effects similar to mecamylamine. Additionally, the dopamine receptor agonist apomorphine (0.03, 0.1, 0.3 mg/kg, s.c.) inhibited naloxone-induced place aversion in morphine-treated rats. CONCLUSION: The inhibitory effect of nicotine on place aversion induced by naloxone-precipitated morphine withdrawal may involve a dopaminergic portion of the central nervous system.

Influence of histamine, cimetidine and pyrilamine on naloxone-induced jumping in morphine-dependent mice.

In the present study, the effects of histamine on naloxone-induced jumping in the presence or absence of adrenoceptor or acetylcholine receptor antagonists in morphine-dependent mice were examined. In these experiments, the drugs were used before s.c. injection of naloxone (2 mg/kg), to test their effects on the expression of jumping. The i.c.v. administration of histamine (5-20 microg/mouse) 15 min before naloxone injection decreased the number of jumps in mice. When the histamine H(2) receptor antagonist, cimetidine (5-20 mg/kg), and the histamine H(1) receptor antagonist, pyrilamine (5-20 mg/kg), were administered i.p. to morphine-dependent mice, only cimetidine enhanced the jumping behaviour. Administration of cimetidine (20 mg/kg, i.p.), 30 min, of the beta-adrenoceptor antagonist, propranolol (2.5-10 mg/kg, i.p.), 15 min but not of pyrilamine (20 mg/kg, i.p.), 30 min before naloxone injection, decreased the histamine effect. The i.p. administration of an acetylcholine receptor antagonist, atropine (5 and 10 mg/kg, i.p.), the alpha(1)-adrenoceptor antagonist, prazosin (0.5, 1 and 2 mg/kg, i.p.), and alpha(2)-adrenoceptor antagonist, yohimbine (0.5, 1 and 2 mg/kg, i.p.), 15 min before naloxone injection, had no effect on the histamine response. Single administration of propranolol, atropine or prazosin decreased, while yohimbine increased the naloxone-induced jumping. It is concluded that the histamine H(2) receptor mechanism may be involved in the influence of histamine on the expression of naloxone-induced jumping in morphine-dependent mice.