Antinociceptive effects of central administration of the endogenous cannabinoid receptor type 1 agonist VDPVNFKLLSH-OH [(m)VD-hemopressin(α)], an N-terminally extended hemopressin peptide.

The cannabinoid system has been demonstrated to modulate the acute and chronic pain of multiple origins. Mouse VD-hemopressin(α) [(m)VD-Hpα], an 11-residue α-hemoglobin-derived peptide, was recently reported to function as a selective agonist of the cannabinoid receptor type 1 (CB1) in vitro. To characterize its behavioral and physiological properties, we investigated the in vivo effects of (m)VD-Hpα in mice. In the mouse tail-flick test, (m)VD-Hpα dose-dependently induced antinociception after supraspinal (EC50 = 6.69 nmol) and spinal (EC50 = 2.88 nmol) administration. The antinociceptive effects of (m)VD-Hpα (intracerebroventricularly and intrathecally) were completely blocked by N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3- carboxamide (AM251; CB1 antagonist), but not by 6-iodo-2-methyl-1-[2-(4-morpholinyl)ethyl]-1H-indol-3-yl(4-methoxyphenyl)-methanone (AM630; CB2 antagonist) or naloxone (opioid antagonist), showing its selectivity to the CB1 receptor. Furthermore, the central nervous system (CNS) effects of (m)VD-Hpα were evaluated in body temperature, locomotor activity, tolerance development, reward, and food intake assays. At the highly antinociceptive dose (3 × EC50), (m)VD-Hpα markedly exerted hypothermia and hypoactivity after supraspinal administration. Repeated intracerebroventricular injection of (m)VD-Hpα resulted in both development of tolerance to antinociception and conditioned place aversion. In addition, central injection of (m)VD-Hpα dose-dependently stimulated food consumption. These findings demonstrate that this novel cannabinoid peptide agonist induces CB1-mediated central antinociception with some CNS effects, which further supports a CB1 agonist character of (m)VD-Hpα. Moreover, the current study will be helpful to understand the in vivo properties of the endogenous peptide agonist of the cannabinoid CB1 receptor.

Naloxone blocks the beneficial effects of aqueous extract of Murraya koenigii (L.) Spreng leaves in models of pain.

AIM: This study investigated the antinociceptive effects of aqueous extract of Murraya koenigii (AEMK) leaves (200, 400 and 800 mg/kg, orally) on animal models of acute and persistent pain and its modulation by naloxone. MATERIALS AND METHODS: Antinociceptive effects were assessed using tail-flick, hot plate and formalin tests in mice. To differentiate between central and peripheral antinociceptive effect of AEMK, naloxone (2 mg/kg) was administered along with the 800 mg/kg dose of extract. Morphine was used as a standard drug. RESULTS: AEMK and morphine significantly increased the tail-flick latency (tfl) and paw licking/jumping latency in tail-flick and hot plate tests, respectively, in comparison to control. Also, in both the tests AEMK and morphine significantly increased the AUC0-120 min. In formalin test, AEMK (400 mg/kg and 800 mg/kg) and morphine significantly reduced licking time in both early and late phases in comparison to control. CONCLUSIONS: Thus, in all three pain models AEMK showed antinociceptive effect, which was blocked by naloxone suggesting the involvement of opioidergic central mechanism.

Effects of cannabinoid agonists on sheep sphincter of oddi in vitro.

BACKGROUND/AIMS: According to recent studies, the endocannabinoid system plays an important role in both physiological and pathophysiological situations. The purpose of the present study was to investigate the effects of cannabinoid (CB) agonists on isolated sheep sphincter of Oddi (SO)in vitro. METHODS: The isolated sheep SO tissues were mounted in organ baths and tested for isometric tension and cyclic GMP levels (cGMP) in response to the non-selective CB receptor agonist WIN 55,212-2 and the potent CB1 receptor agonist methanandamide in the presence and absence of the selective CB1 antagonist SR 141716A, the selective CB2 antagonist SR 144528 and the nonspecific inhibitor of nitric oxide (NO) synthase L-NAME. RESULTS: CB agonists relaxed SO in a concentration-dependent manner. These relaxations did not reduce in the presence of SR 144528 but were significantly reduced by SR 141716A and L-NAME. Carbachol significantly increased the cGMP levels compared with the control group and both of the CB receptor agonists significantly increased the cGMP levels compared with the control and carbachol groups. On the other hand, L-NAME prevented the increase in cGMP levels caused by CB agonists. CONCLUSION: These results show that the relaxation by the agonists may be through CB1 receptors. The decrease of CB relaxation responses by L-NAME, a nonspecific inhibitor of NO synthase, and the increase of cGMP levels in the SO tissues by CB agonists which decreased by L-NAME show that the relaxation effects of these agonists may also partially be via increasing the NO synthesis or release.

Antioxidant and antinociceptive effects of Citrus limon essential oil in mice.

The antioxidant and antinociceptive activities of Citrus limon essential oil (EO) were assessed in mice or in vitro tests. EO possesses a strong antioxidant potential according to the scavenging assays. Moreover, it presented scavenger activity against all in vitro tests. Orally, EO (50, 100, and 150 mg/kg) significantly reduced the number of writhes, and, at highest doses, it reduced the number of paw licks. Whereas naloxone antagonized the antinociceptive action of EO (highest doses), this suggested, at least, the participation of the opioid system. Further studies currently in progress will enable us to understand the action mechanisms of EO.

Physiological basis for inhibition of morphine and improgan antinociception by CC12, a P450 epoxygenase inhibitor.

Many analgesic drugs, including µ-opioids, cannabinoids, and the novel nonopioid analgesic improgan, produce antinociception by actions in the rostral ventromedial medulla (RVM). There they activate pain-inhibiting neurons, termed "OFF-cells," defined by a nociceptive reflex-related pause in activity. Based on recent functional evidence that neuronal P450 epoxygenases are important for the central antinociceptive actions of morphine and improgan, we explored the convergence of opioid and nonopioid analgesic drug actions in RVM by studying the effects of the P450 epoxygenase inhibitor CC12 on the analgesic drug-induced activation of these OFF-cells and on behavioral antinociception. In rats lightly anesthetized with isoflurane, we recorded the effects of intraventricular morphine and improgan, with and without CC12 pretreatment, on tail flick latency and activity of identified RVM neurons: OFF-cells, ON-cells (pronociceptive neurons), and neutral cells (unresponsive to analgesic drugs). CC12 pretreatment preserved reflex-related changes in OFF-cell firing and blocked the analgesic actions of both drugs, without interfering with the increase in spontaneous firing induced by improgan or morphine. CC12 blocked suppression of evoked ON-cell firing by improgan, but not morphine. CC12 pretreatment had no effect by itself on RVM neurons or behavior. These data show that the epoxygenase inhibitor CC12 works downstream from receptors for both µ-opioid and improgan, at the inhibitory input mediating the OFF-cell pause. This circuit-level analysis thus provides a cellular basis for the convergence of opioid and nonopioid analgesic actions in the RVM. A presynaptic P450 epoxygenase may therefore be an important target for development of clinically useful nonopioid analgesic drugs.

Enterostatin (APGPR) suppresses the analgesic activity of morphine by a CCK-dependent mechanism.

Enterostatin (APGPR) found in the gastrointestinal tract and brain is an anorectic pentapeptide. We found that APGPR inhibited morphine-induced analgesia after intracerebroventricular administration in mice at a dose of 10nmol/mouse. The anti-analgesic effect of APGPR was inhibited by pretreatment with lorglumide and LY225910, antagonists for cholecystokinin 1 (CCK1) and cholecystokinin 2 (CCK2) receptors, respectively. The anti-analgesic effect of APGPR may be mediated by CCK release, since APGPR does not have affinity for CCK receptors.

In vivo characterization of (-)(-)MCL-144 and (+)(-)MCL-193: isomeric, bivalent ligands with mu/kappa agonist properties.

Once opioid receptor dimers were postulated, a goal has been to synthesize and screen novel opioids, with the hope of furthering our knowledge of the structure-activity relationship of opioid ligands with the opioid receptors. The aim of the current study was to address whether two isomeric bivalent ligands would have pharmacological differences after central administration, in vivo. The two compounds, (-) bis(N-cyclobutylmethyl-morphinan-3-yl) sebacoylate dihydrochloride (MCL-144) and 1-((+)N-cyclobutylmethylmorphinan-3-yl)-10-((-) N-cyclobutylmethylmorphinan-3-yl)sebacolyate (MCL-193) are each linked by a 10-carbon chain ester. The active (-) enantiomer for both ligands is 3-hydroxy-N-cyclobutylmethyl morphinan ((-)MCL-101), a N-cyclobutylmethyl analogue of cyclorphan (J Med Chem 43:114-122, 2000). MCL-144 contains two active levo rotatory (-)(-) pharmacophores, while MCL-193 contains one active (-) and one inactive (+) pharmacophore of MCL-101. In vitro analysis demonstrated that all three compounds, (-)(-)MCL-144, (+)(-)MCL-193 and (-)MCL-101 were kappa agonists and mu partial agonists. (-)(-)MCL-144 and (-)MCL-101 had much higher affinity for both the mu and kappa opioid receptors compared to (+)(-)MCL-193. In vivo, (-)(-)MCL-144 and (+)(-)MCL-193 produced full dose-response curves, in the 55 degrees C tail-flick test, with each compound having an ED(50) value of 3.0 nmol after intracerebroventricular (i.c.v.) administration. The analgesic properties of both compounds were antagonized by the mu-selective antagonist, beta-funaltrexamine and the kappa-selective antagonist nor-binaltorphimine. Concomitant, i.c.v., administration of either (-)(-)MCL-144 or (+)(-)MCL-193 with morphine, did not significantly antagonize morphine-induced antinociception at any dose tested. In antinociceptive tests, (-)(-)MCL-144 and (+)(-)MCL-193 had the same pharmacological properties, demonstrating that having two active pharmacophores separated by a 10-carbon spacer group did not increase the antinociceptive efficacy of the compound. Additionally, it was also of interest to compare (-)(-)MCL-145 and (-)(-)MCL-144, as the only difference between these bivalent ligands is the spacer region connecting the two pharmacophores, yet (-)(-)MCL-145 produced an ED(50) value 10-fold lower than (-)(-)MCL-144 (ED(50) values = 0.3 nmol and 3.0 nmol, respectively).

Midazolam attenuates the antinociception induced by d-serine or morphine at the supraspinal level in rats.

Our recent study has shown that the intracerebroventricular administration of d-serine, an endogenous and selective agonist for the glycine site of the N-methyl-d-aspartate receptor, alone or in combination with morphine, leads to the potentiation of antinociception on the tail-flick response. Although there is a variety of information concerning the effects of benzodiazepines on opioid-induced antinociception, little is known about the effect of benzodiazepines on the N-methyl-d-aspartate receptor agonist-induced antinociception. To clarify the analgesic interactions among the benzodiazepine/GABA(A), N-methyl-d-aspartate and opioid receptors at the supraspinal level, we investigated the effects of intracerebroventricular administration of midazolam, a benzodiazepine receptor agonist, on the antinociception evoked by the intracerebroventricular application of d-serine or morphine. The intracerebroventricular administration of midazolam alone produced hyperalgesia on the tail-flick response in a benzodiazepine receptor antagonist, flumazenil-reversible manner. The antinociception induced by the intracerebroventricular application of d-serine or morphine was attenuated by the intracerebroventricular administration of midazolam. In addition, this inhibitory effect of midazolam on the antinociception of d-serine or morphine was antagonized by the intracerebroventricular administration of flumazenil. Together with the facts that d-serine and midazolam act as selective agonists for the glycine site of the N-methyl-d-aspartate receptor and benzodiazepine/GABA(A) receptor, respectively, these observations suggest a functional interaction between the NMDA and benzodiazepine/GABA(A) receptors in the regulation of antinociception at the supraspinal level.

Modulation by female sex hormones of the cannabinoid-induced catalepsy and analgesia in ovariectomized mice.

Cannabinoids are psychoactive compounds with many pharmacological properties such as analgesia, sedation and catalepsy most of which are mediated by cannabinoid CB1 receptors. In the present study, we evaluated whether the ovarian sex hormones are involved in the cannabinoid-induced catalepsy and analgesia in ovariectomized female mice. Female NMRI mice (weighing 25-30 g) were divided into 3 main groups: unoperated, sham-operated and ovariectomized. Both the catalepsy and analgesia induced by different doses of the synthetic cannabinoid WIN 55,212-2 (2 and 4 mg/kg, i.p.) were examined in the groups in the presence or absence of the cannabinoid CB1 antagonist AM251 (0.5 mg/kg). We also evaluated effects of estradiol valerate (10 mg/kg) and progesterone (25 mg/kg) on catalepsy and analgesia induced by WIN 55,212-2 in ovariectomized mice. The antinociceptive effect of WIN 55,212-2 was significantly (P<0.01) enhanced in ovariectomized mice, which was prevented by pretreatment with estradiol but not by progesterone. There was no significant difference in the cannabinoid-induced catalepsy between control and ovariectomized mice. However, pretreatment with progesterone but not estradiol potentiated the cataleptic effect of low dose of WIN 55,212-2 (2 mg/kg) in ovariectomized mice (P<0.01). The present data demonstrated for the first time that ovarian sex steroids could modulate both cannabinoid-induced catalepsy and analgesia in female ovariectomized mice.

Activation of opioid receptor like-1 receptor in the spinal cord produces sex-specific antinociception in the rat: estrogen attenuates antinociception in the female, whereas testosterone is required for the expression of antinociception in the male.

Sex-related differences in the perception and modulation of pain have been reported. The present study is the first to investigate systematically whether activation of opioid receptor-like 1 receptor (ORL1) by orphanin FQ (OFQ) produces sex-specific modulation of spinal nociception and whether estrogen or testosterone contributes to these differences using the rat as an experimental animal. Two behavioral models, the NMDA and heat-induced nociceptive tests, were used to examine sex-specific modulation of spinal nociception. Intrathecal microinjection of OFQ in male, ovariectomized (OVX), and diestrous rats produced a significant antinociceptive effect on both tests. However, OFQ failed to produce antinociception in proestrous rats, the phase of the estrous cycle with the highest levels of circulating estradiol, and produced a dose-dependent effect in OVX females treated with 1 ng to 100 microg of estradiol. The antinociceptive effects of OFQ were dose dependent in male and OVX animals and were reversibly antagonized by UFP-101 ([Nphe1,Arg14,Lys15]N/OFQ(1-13)-NH2), an ORL1 receptor-selective antagonist. Interestingly, OFQ was ineffective in gonadectomized (GDX) males, whereas testosterone replacement restored the antinociceptive effect of OFQ in GDX males. We conclude that OFQ produces sex-specific modulation of spinal nociception; estrogen attenuates antinociception in the female in parallel with normal cycling of estrogen levels, and testosterone is required for the expression of antinociception in the male; thus, the sensitivity of the male to the antinociceptive effects of OFQ is not simply attributable to the intrinsically low estrogen levels in these animals.

Involvement of mu- and delta-opioid receptors in the antinociceptive effects induced by AMPA receptor antagonist in the spinal cord of rats.

The present study was performed to explore the involvement of opioid receptors in the antinociception induced by a-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor antagonist in rats. The hindpaw withdrawal latency (HWL) to noxious thermal and mechanical stimulation was assessed by hot plate test and the Randall Selitto Test. Intrathecal injection of 20 nmol of 1,2,3,4-tetrahydro-6-nitro-2,3-dioxo[f]quinoxaline-7-sulfonamide (NBQX) disodium, a competitive AMPA receptor antagonist, increased significantly the HWLs to both thermal and mechanical stimulation in rats. The increased HWLs induced by NBQX were dose-dependently attenuated by the opioid receptor antagonist naloxone, while naloxone itself had no marked influences on the HWL of rats. Furthermore, the increased HWLs induced by NBQX were inhibited by the mu-opioid antagonist beta-funaltrexamine (beta-FNA) or the delta-opioid antagonist naltrindole, but not by the kappa-opioid antagonist nor-binaltorphimine (nor-BNI). The results suggest that mu- and delta-opioid receptors, not kappa-opioid receptor, are involved in the antinociception induced by AMPA antagonist in the spinal cord of rats.

Role of the opioidergic system and nitric oxide in the analgesic effect of venlafaxine.

The noradrenalin and serotonin re-uptake inhibitor venlafaxine has an analgesic effect that is independent of its antidepressant activity; however, the mechanism of this effect remains to be elucidated. This study was performed to investigate the possible roles of the opioidergic system and nitric oxide (NO) pathway in the analgesic effect of venlafaxine. Eighty Wistar rats of both sexes were allocated to 10 groups. The hot plate test was used to assess the antinociceptive/analgesic effect. The temperature of the hot plate was adjusted to 52.5+/-1 degrees C, the cut-off period was set to be 50 sec; licking of the hind paw was used as a sign of pain perception. Venlafaxine alone (25 mg/kg) showed marked analgesic activity (p<0.05). N-omega-nitro-L-arginine (L-NOARG) alone (20 mg/kg) and naloxone alone (2 mg/kg and 4 mg/kg) showed no analgesic activity (p>0.05). Coadministration of low-dose naloxone (2 mg/kg) and both doses of L-NOARG (20 and 40 mg/kg) with venlafaxine (25 mg/kg) did not modify the analgesic effect but high-dose naloxone (4 mg/kg) decreased it significantly (p<0.05). In conclusion, these results suggest that the opioidergic system but not the NO pathway has a role in the analgesic effect of venlafaxine.

Attenuation of nicotine-induced antinociception, rewarding effects, and dependence in mu-opioid receptor knock-out mice.

The involvement of mu-opioid receptors in different behavioral responses elicited by nicotine was explored by using mu-opioid receptor knock-out mice. The acute antinociceptive responses induced by nicotine in the tail-immersion and hot-plate tests were reduced in the mutant mice, whereas no difference between genotypes was observed in the locomotor responses. The rewarding effects induced by nicotine were then investigated using the conditioning place-preference paradigm. Nicotine produced rewarding responses in wild-type mice but failed to produce place preference in knock-out mice, indicating the inability of this drug to induce rewarding effects in the absence of mu-opioid receptors. Finally, the somatic expression of the nicotine withdrawal syndrome, precipitated in dependent mice by the injection of mecamylamine, was evaluated. Nicotine withdrawal was significantly attenuated in knock-out mutants when compared with wild-type mice. In summary, the present results show that mu-opioid receptors are involved in the rewarding responses induced by nicotine and participate in its antinociceptive responses and the expression of nicotine physical dependence.

Inhibitors of serine/threonine protein phosphatases antagonize the antinociception induced by agonists of alpha 2 adrenoceptors and GABAB but not kappa-opioid receptors in the tail flick test in mice.

We previously reported that serine/threonine protein phosphatases (PPs) play a role in the antinociception induced by the mu-opioid receptor agonist morphine. In this study we evaluated the possible involvement of PPs on the antinociception induced by agonists of others G protein-coupled receptors in the tail flick test in mice. The subcutaneous administration of clonidine (0.25-4 mg/kg), baclofen (2-32 mg/kg) or U50,488H (2-16 mg/kg) (agonists of alpha(2) adrenoceptors, GABA(B) and kappa-opioid receptors, respectively) produced dose-dependent antinociception. The antinociceptive effects of clonidine and baclofen were antagonized in a dose-dependent way by the protein phosphatase inhibitors okadaic acid (0.001-10 pg/mouse, i.c.v.) and cantharidin (0.001-10 ng/mouse, i.c.v.), and okadaic acid was 1000 times more potent than cantharidin in producing this effect. The effects of these drugs appear to be specifically due to the blockade of PPs, since L-norokadaone (an analogue of okadaic acid that has no effect on PPs) did not modify clonidine- or baclofen-induced antinociception over the wide range of doses used (0.001-1000 pg/mouse, i.c.v.). On the other hand, the antinociception induced by activation of kappa-opioid receptors with U50,488H was not modified by okadaic acid or cantharidin. In conclusion, our data support the idea that serine/threonine PPs are differentially involved in the antinociceptive effects of several agonists of G protein-coupled receptors in mice.

Nociceptin/orphanin FQ prevents the antinociceptive action of paracetamol on the rat hot plate test.

Nociceptin/orphanin FQ (N/OFQ) is involved in many behavioural patterns; in particular, it exerts a modulating effect on nociception. Like other proposed antiopiates, nociceptin/orphanin FQ has been shown to have analgesic, hyperalgesic as well as antianalgesic properties. Among the various effects proposed on nociceptive sensitivity at supraspinal level, the antagonistic activity toward morphine analgesia seems to be of interest. Therefore, we decided to investigate whether nociceptin/orphanin FQ and [Arg14, Lys15] nociceptin/orphanin FQ (R-K, a nociceptin analogue) can have the same effect on the analgesia produced by nonopioid analgesics. In this study, we examined the antianalgesic effect of nociceptin/orphanin FQ and its analogue R-K on paracetamol-induced analgesia and evaluated by means of the hot plate test in rats. Nociceptin/orphanin FQ was intracerebroventricularly administered, and, after 5 min, a dose of 400 mg/kg paracetamol was injected intraperitoneally, 30 min before the hot plate test. Nociceptin/orphanin FQ and R-K showed a dose-dependent antagonism on the antinociceptive effect of paracetamol, and the activity of both drugs was significantly reduced by the antagonist [Nphe1] Arg14, Lys15-N/OFQ-NH2 (UFP-101). These data indicate that nociceptin/orphanin FQ and R-K have an antianalgesic effect on the analgesia produced by a nonopioid analgesic drug, like paracetamol, that seems to develop within the brain.

Pharmacologically selective block of mu opioid antinociception by peptide nucleic acid antisense in absence of detectable ex vivo knockdown.

The goal of this study was to determine the neuroanatomical extent of mu opioid receptor knockdown in central nervous system (CNS) following intracerebroventricular (i.c.v.) administration of peptide nucleic acid antisense. Rats received subchronic i.c.v. injections of anti-mu opioid receptor antisense, mismatch or vehicle, and were tested for paw pressure latency following i.c.v. mu opioid receptor agonist ([D-Ala2, N-Me-Phe4, Gly-ol5]-enkephalin; DAMGO) or delta opioid receptor agonist ((+)-4-[(aR)-a-((2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl]-N,N-diethylbenzamide; SNC80). The anti-mu opioid receptor antisense (but not mismatch) sequence abolished DAMGO-induced antinociception with no reduction in the delta opioid receptor-mediated response. In contrast, postmortem receptor autoradiographic analysis of CNS areas revealed no change in mu opioid receptor functional response ([35S]GTPgammaS assay) or receptor labelling ([125I]FK-33824 and mu opioid receptor immunoautoradiography). These results provide further evidence for antisense-induced knockdown at the behavioural level in the absence of clear changes at the tissue level.

In vivo characterization of a novel inhibitor of CNS nicotinic receptors.

There are multiple types of nicotine acetylcholine receptors (nAChR) in the brain associated with synaptic function, signal processing, or cell survival. The therapeutic targeting of nicotinic receptors in the brain will benefit from the identification of drugs, which may be selective for their ability to activate or inhibit a limited range of these receptor subtypes. We previously identified a family of bis-tetramethylpiperidine compounds as selective inhibitors of neuronal-type nicotinic receptors. In the present study we describe the in vivo effects and properties of 2,2,6,6-tetramethylpiperidin-4-yl heptanoate (TMPH), a novel inhibitor of neuronal nicotinic receptors. Delivered systemically, this drug can block central nervous system effects of nicotine, indicating that this drug is able to cross the blood-brain barrier and access sites in the brain. Unlike the prototype CNS-active nicotinic inhibitor, mecamylamine, TMPH blocked some but not all of the CNS effects of nicotine, indicating that it has a unique selectivity for specific receptor subtypes in the brain. The nAChR subtypes that mediate the locomotor effects and hypothermic effects of nicotine appear to be less sensitive to TMPH than those which mediate analgetic effects and discriminative stimuli. These results indicate that TMPH may possess unique selectivity for specific nicotinic receptor subtypes.

Morphine-3-glucuronide may functionally antagonize morphine-6-glucuronide induced antinociception and ventilatory depression in the rat.

The effects of the major morphine metabolites, morphine-3-glucuronide and morphine-6-glucuronide, on nociception were assessed by the tail-flick, hot-plate and writhing tests in the rat. Morphine-3-glucuronide (M3G) 1.1 x 10(-9) mol (0.5 micrograms) or saline was injected intracerebroventricularly (i.c.v.) or intrathecally (i.t.) followed by a second injection of 2.0 x 10(-10) mol (0.1 microgram) or 2.0 x 10(-11) mol (0.01 microgram) morphine-6-glucuronide (M6G) 10 min later. Administration of M3G (i.c.v.) significantly attenuated the antinociceptive effects of M6G in the hot-plate test. After i.t. administration, the antinociceptive effect of M6G in all three tests was significantly reduced in the M3G pretreated group compared to the group receiving saline. The ventilatory effects of 4.0 x 10(-9)-1.0 x 10(-8) mol (2-5 micrograms) M6G and 1.7-2.2 x 10(-8) mol (8-10 micrograms) M3G given i.c.v. were studied by a whole-body plethysmographic technique in halothane anaesthetized rats. Separate groups of rats received M3G followed by M6G injection or vice versa. In animals receiving M3G there was a prevention or attenuation of the M6G induced depression of respiratory frequency, tidal volume and minute ventilation compared to control groups receiving M6G in combination with saline. These results show that M3G may functionally antagonize the central antinociceptive effects as well as the ventilatory depression induced by M6G. Interestingly, M3G was more potent in antagonizing the M6G-induced analgesia after i.t. administration than that after i.c.v. administration, which may suggest that the spinal cord is more sensitive to the non-opioid excitatory effects of M3G than supraspinal structures.

Rat knee-joint carrageenin incapacitation test: an objective screen for central and peripheral analgesics.

A new behavioral test is described in which quantitation is independent of the observer and is sensitive to all classes of analgesics. A computer-assisted device measures the period during which a rat hind paw fails to touch the surface of a rotating cylinder for 1 min (paw elevation time). Intra-articular injection of carrageenin induces a progressive and dose-dependent incapacitation of the limb. The maximum paw elevation time is attained 3-4 h after carrageenin challenge. The model showed dose-dependent sensitivity to (a) a central acting opiate (morphine, ID50 = 1.5 mg/kg, i.p.), (b) cyclooxygenase inhibitors (indomethacin, ID50 = 0.8 mg/kg, i.p.; diclofenac, ID50 = 0.22 mg/kg, i.p.), and (c) peripheral analgesics which directly antagonize nociceptor hypersensitivity: dipyrone (ID50 = 21 mg/kg, i.p.), N-methyl-nalorphine (ID50 = 14 mg/kg, i.p.) and BW443C (ID50 = 17.5 mg/kg, i.p.). The knee-joint carrageenin incapacitation was also blocked by the sympatholytics, propranolol and guanethidine. After the blockade by either indomethacin or guanethidine, intra-articular injections of prostaglandin E2 or dopamine, respective, reversed carrageenin-induced incapacitation. These results suggest that during inflammatory articular incapacitation cyclooxygenase and sympathomimetic mediators are involved, as has been suggested for the rat paw carrageenin hyperalgesia test and formalin test.

Involvement of endogenous beta-endorphin in antinociception in the arcuate nucleus of hypothalamus in rats with inflammation.

Although exogenous administration of beta-endorphin to the arcuate nucleus of hypothalamus (ARC) had been shown to produce antinociception, the role of endogenous beta-endorphin of the ARC in nociceptive processing has not been studied directly. The aim of the present study was to investigate the effect of endogenous beta-endorphin in the ARC on nociception in rats with carrageenan-induced inflammation. The hindpaw withdrawal latency (HWL) to noxious thermal and mechanical stimulation was assessed by the hot-plate test and the Randall Selitto Test. Intra-ARC injection of naloxone had no significant influence on the HWL to thermal and mechanical stimulation in intact rats. The HWL decreased significantly after intra-ARC injection of 1 or 10 microg of naloxone in rats with inflammation, but not with 0.1 microg of naloxone. Furthermore, intra-ARC administration of the selective mu-opioid receptor antagonist beta-funaltrexamine (beta-FNA) decreased the nociceptive response latencies to both stimulation in a dose-dependent manner in rats with inflammation, while intra-ARC administration of the selective delta-opioid receptor antagonist naltrindole or the selective kappa-opioid receptor antagonist nor-binaltorphimine (nor-BNI) showed no influences on the nociceptive response latency. The antiserum against beta-endorphin, administered to the ARC, also dose-dependently reduced the HWL in rats with inflammation. The results indicate that endogenous beta-endorphin in the ARC plays an important role in the endogenous antinociceptive system in rats with inflammation, and that its effect is predominantly mediated by the mu-opioid receptor.