Spinal antinociceptive synergy between clonidine and morphine, U69593, and DPDPE: isobolographic analysis

The spinal antinociceptive interaction between the opiate receptor subtype agonists morphine (mu), U69593 (kappa) and [D-Pen2,5]-enkephalin (DPDPE; delta) with clonidine (alpha 2 adrenergic) was examined. Male SD rats received fixed ratios of clonidine to morphine (10:1), U69593 (1:3), or DPDPE (10:1) through catheters terminating at the lumbar cord. Graded dose-response curves (DRC) were constructed from tail-flick latencies converted to % maximal possible effect (%MPE), and the ED50 calculated. The DRCs of morphine and U69593 but not of DPDPE were parallel to the DRC of the opiate plus clonidine. Synergy was determined by isobolographic analysis. The ED50 values for the mixtures were significantly less than the theoretical additive ED50 values, indicating synergy between clonidine and morphine, U69593, or DPDPE.     

Modulation of morphine induced antinociception by intracerebroventricularly administered captopril

Captopril when administered intracerebroventricularly (icv) in doses of 100, 300, 500 and 1000 micrograms induced a dose dependent antinociceptive effect in rats. Naloxone pretreatment (10 mg/kg, ip) completely antagonised antinociceptive effect of captopril, suggesting thereby the involvement of brain enkephalinergic system. Captopril 300 micrograms, icv potentiated the antinociceptive effect of morphine in intact animals. The bilateral adrenalectomy did not have any effect on this potentiation as against the reported blockade of potentiation in adrenalectomized animals when captopril was administered by systemic route. Thus potentiation of morphine induced antinociception by icv captopril is unlikely to be exerted through an effect on adrenal function and is most likely due to increased brain enkephalin levels.     

Behavioral responses to intracerebroventricularly administered neurohypophyseal peptides in mice

Lysine vasopressin (LVP), arginine vasopressin, oxytocin, and arginine vasotocin administered intraventricularly (icv) to mice all provoked a dose-dependent behavioral response in the range 0.1 – 1.0 μg. This response included a pronounced hyperactivity, extensive foraging, increased grooming, and at higher doses, stereotyped scratching, squeaking, and occasional barrel rolling. The four hormones were all approximately equipotent. Desglycinamide lysine vasopressin and [desaminocys₁, D-Arg₈] vasopressin produced some of the characteristic behaviors, but were much less potent. While pretreatment of the animals with reserpine (5 mg/kg ip), haloperidol (0.5 mg/kg ip), or physostigmine (0.5 mg/kg ip) sedated the animals and attenuated the locomotion and grooming, these drugs did not substantially alter the characteristic behavioral responses to LVP. Pretreatment with α-methyl-p-tyrosine (400 mg/kg ip), p-chlorophenylalanine (320 mg/kg ip), 6-hydroxydopamine (100 μg icv), ergotamine (0.5 μg icv), ethoxolamide (52 ng icv), diphenhydramine (20 μg icv), prostaglondin F_2α (2 μg icv), or naloxone (1 mg/kg ip) did not alter the LVP-induced behaviors. None of these drugs or -amphetamine (0.5 to 20 mg/kg ip) or nicotine (0.1 or 1 μg icv) mimicked the behavioral effects of the hormones.     

The occurrence of cross-tolerance between morphine and ethyl-ketocyclazocine using the tail-flick test: Lack of effect of diazepam,phenobarbital, or amphetamine

Experiments were designed to test for short-term tolerance to morphine and ethyl-ketocyclazocine (EKC), mu and kappa agonists, respectively, and cross-tolerance between the two drugs. Mice were primed with one of the drugs, using doses that did not affect the tail-flick response when tested at a time 1 or 3 hours later, when the same or alternate test drug was administered. All animals were injected with the priming drug IP. In one series of experiments, the test drugs were given SC, and in the other, the test drugs were injected ICV under brief halothane anesthesia. Priming with morphine (30 or 100 mg/kg) significantly raised the ED₅₀ for ICV morphine. Priming with EKC (2 or 6 mg/kg) similarly elevated the ED₅₀'s for SC and ICV EKC. Symmetrical cross-tolerance was produced in experiments where the test drugs were administered SC when tested at 3 hrs. The effects of priming with EKC on morphine analgesia was evident when the interval between priming and test drugs was 1 hour. When the test drugs were given ICV, cross-tolerance was also symmetrical: priming with EKC significantly raised the ED₅₀ for morphine and priming with morphine raised the ED₅₀ for EKC when tested at 3 hrs. These data suggest that both agonists act on a common site to produce analgesia as similar pA₂ values for naloxone antagonism were determined. The occurrence of short-term tolerance and cross-tolerance to the opiates was unaltered by chronic pretreatment with diazepam, phenobarbital, or amphetamine.     

Fangchinoline inhibited the antinociceptive effect of morphine in mice

Fangchinoline (FAN), a non-specific calcium antagonist, is a major alkaloidal component of the creeper Stephania tetrandra S. Moore (or fenfangji). It has been shown to possess antagonistic activity on morphine-induced antinociception in mice. This study was undertaken to assess the antagonistic mechanism. The results demonstrated that FAN (IP) attenuated morphine (SC)-induced antinociception in a dose-dependent manner with significant effect at doses of 30 and 60mg/kg body wt. (IP) in the tail-flick test but not the tail-pinch tests, carried out in mice. This antagonism was abolished by pretreatment with a serotonin precursor, 5-hydroxytryptophan (5-HTP, IP), but not by pretreatment with a noradrenaline precursor, L-dihydroxyphenylalanine (L-DOPA, IP) in the tail-flick test. On the other hand, the development of morphine-induced analgesic tolerance was not prevented by FAN. These results suggest that the serotonergic pathway may be involved in the antagonism of morphine-induced antinociception by FAN and, in agreement with other reports, also indicates the possible dissociation of the morphine analgesic effect from its tolerance-development mechanism.     

Dynorphin (1-13): analgesia, hypothermia, cross-tolerance with morphine and beta-endorphin

Intracerebroventricular administration of 20, 40 and 60 nmol of dynorphin (1-13) produced analgesia, as assessed by flinch/jump response to footshock, and hypothermia in the rat. Rats developed tolerance to both the analgesic and thermic effects of the 20 nmol dose of dynorphin. Dynorphin and beta-endorphin showed cross-tolerance with respect to their analgesic but not their thermic effects. Dynorphin and morphine also produced cross-tolerant analgesic effects. Naloxone (10 mg/kg, IP) completely blocked the barrel rolling produced by 20 nmol dynorphin but did not alter its analgesic or thermic effects.     

Environmental and intraperitoneal ethanol influences morphine antinociceptive effect in mice

The ability of acute environmental or intraperitoneal (i.p.) ethanol to influence morphine antinociceptive effect was studied in mice. In order to induce tolerance to morphine analgesia, mice received daily injections of 10 mg/Kg morphine over a period of 10 days. Mice were divided into three groups: i.p. ethanol (E), environmental ethanol (E*), and control saline (M). During the induction of tolerance these groups were treated identically except on days 1 and 11. On these days, 10 minutes prior to morphine injection, mice received either i.p. ethanol (1g/Kg), environmental ethanol (a bottle of 10% ethanol placed next to the animals cage during the experiments), or an equivalent volume of saline. Analgesia was assessed using a standard hot plate protocol and dose-response cumulative curves for morphine analgesia were obtained on days 1 and 11. On day 1, both the i.p. and environmental administration of ethanol showed similar morphine-potentiation effects [Mean Effective Dose: ED50 (M1)=4.5 mg/kg; ED50 (E1)=2.4 mg/kg; ED50 (E*1)=2.1 mg/kg]. On day 11, control group mice showed a reduction of morphine analgesia at test [ED50 (M11)=14.1 mg/kg]. Mice receiving i.p. and environmental ethanol again showed a leftward shift in dose-response cumulative curves for morphine antinociception with respect to controls [ED50 (E11)=9.1 mg/kg; ED50 (E*11)=4.7 mg/kg]. I.p. ethanol administration at non-antinociceptive doses enhances the morphine antinociception effect similarly in tolerant and non-tolerant (naive) mice. The presence of environmental ethanol can also induce a similar pattern of increase in morphine antinociception effect.     

Synergy between the antinociceptive effects of morphine and NSAIDs

The intraperitoneal administration of morphine, diclofenac, ketoprofen, meloxicam, metamizol, paracetamol and piroxicam induced dose-dependent antinociception in mice tested with the acetic acid writhing test. The isobolographic analysis of the simultaneous intraperitoneal administration of fractions of the ED50's of morphine with each nonsteroidal anti-inflammatory drug (NSAID) demonstrated the existence of a supra-additive interaction (synergy). The selective antagonist of micro -opioid receptors naltrexone partially reversed the supra-additive interactions to additive interactions; however, the combinations of morphine/metamizol and morphine/paracetamol were completely antagonized, resulting in subadditive interactions. The selective antagonist of delta-opioid receptors naltrindole failed to significantly attenuate the combinations of morphine with ketoprofen, meloxicam and piroxicam, but decreased the activity of the combinations of morphine with diclofenac, metamizol and paracetamol, transforming the interactions from supra-additive to additive. Nor-binaltorphimine was used to evaluate the involvement of kappa-opioid receptors. Nor-binaltorphimine did not modify the supra-additive interaction of morphine and NSAIDs and the additive interaction of the co-administration of morphine and metamizol. The synergy between morphine and NSAIDs could be related to different pathways of pain transmission, probably related to the different intracellular signal transduction mechanisms of action of opioid and non-opioid agents.     

Decreased spinal morphine/clonidine antinociceptive synergism in morphine-tolerant mice

The antinociceptive interactions between spinally administered opioids and the alpha₂ agonist clonidine were examined in placebo and morphine pellet-implanted mice using the tail flick test. In placebo pellet-implanted animals, coadministered morphine and clonidine produced a synergistic antinociceptive effect. In mice implanted with morphine pellets, the synergism decreased to an additive interaction. The interactions between clonidine and the mu agonist Tyr-D-Ala-Gly-N-Me-Phe-Gly-ol (DAMGO), the delta agonist D-Pen²-D-Pen⁵-Enkephalin (DPDPE), and the kappa agonist U50-488H were also synergistic in placebo animals. In morphine pellet treated mice the DPDPE/clonidine interaction decreased to an antagonistic interaction, the DAMGO/clonidine remained synergistic and the U50-488H/clonidine interaction decreased to additive. These results support the proposal that the morphine spinal/supraspinal synergism depends upon the interaction between spinal opioid and alpha₂ receptors and a decrease in this interaction is a mechanism involved in development of tolerance to morphine. In addition, delta and kappa receptors appeared to be more involved in the morphine/clonidine decreased interaction than did mu opioid receptors.     

Differential analgesic cross-tolerance to morphine between lipophilic and hydrophilic narcotic agonists

Mice were rendered tolerant to morphine by the subcutaneous implantation of one 75 mg morphine pellet. Seventy-two hours post-pellet implantation, the animals were evaluated in the tail-flick assay for analgestic tolerance and cross-tolerance to subcutaneously administered morphine, normorphine, methadone, etorphine and intracerebroventricularly administered morphine. With the pellet remaining in situ during testing, there was the expected analgestic tolerance to peripherally administered morphine and analgesic cross-tolerance to normorphine. However, with the pellet in situ during testing, there was a surprising lack of analgesic tolerance to intracerebroventricularly administered etorphine or methadone. In contrast, removal of the morphine pellet 3 hours prior to the analgesic evaluation apparently unmasked the expression of tolerance and cross-tolerance as evidenced by a three fold, parallel shift to the right of the analgesic dose-response curve for subcutaneously administered etorphine and methadone and a seven fold shift for intracerebroventricularly administered morphine. These data emphasize that a more rigorous evaluation of tolerance development methodologies need be explored and support the suggestion that removal of the morphine-inducing pellet prior to analgesic determinations results in a distinct state of “tolerance” quite different from that observed with the pellet remaining in situ during testing.     

Cellular uptake of intracerebroventricularly administered biotin- or digoxigenin-Labeled antisense oligodeoxynucleotides in the rat

Summary 1. Antisense oligodeoxynucleotides (ODNs) internally labeled with biotin or digoxigenin were injected into the lateral ventricle of rats and the distribution of the labeled ODNs was examined at several timepoints following the intracerebroventricular (icv) injections. The stability of these injected antisense ODNs, which had no backbone modifications, was also studied by performing recovery experiments.2. The most intense labeling was observed near the injection site, in periventricular areas, and in perivascular regions. Many of the labeled cells appeared to be neurons, and both the cytoplasm and the nuclei were stained. The labeled cells were detected 15 min after icv injection, demonstrating that the antisense ODNs were taken up rapidly by cells in the parenchyma. The digoxigeninated antisense ODNs were presented in both the cytoplasmic and the nuclear fractions of rat brain extracts, however, the levels appeared to be much lower in the nuclear fractions.3. Antisense ODNs injected into the lateral ventricle seemed to follow the bulk flow of cerebrospinal fluid (CSF), i.e., from the injection site in the lateral ventricle, through the ventricular system, to the subarachnoid spaces and the perivascular spaces. From the ventricular and perivascular spaces, the antisense ODNs diffused into the extracellular space and were taken up by cells. The full-length digoxigeninated antisense ODNs were detectable within cells after only 15 min, indicating their rapid uptake. In addition, the antisense ODNs appeared to be relatively stable in the brain since the full-length digoxigeninated ODNs were still detectable after 4 hr.     

Pharmacokinetics of an intracerebroventricularly administered antibody in rats

The pharmacokinetics (PK) of an antibody in the brain and the spinal cord is insufficiently understood, which is an obstacle to the discovery of antibody drugs that target diseases in the central nervous system. In this study, we focused on the elimination of IgG from cerebrospinal fluid (CSF) circulating in the brain and the spinal cord in rats, and, to evaluate the influence of CSF bulk flow on the clearance of IgG, also examined the PK of inulin in CSF. To monitor their concentrations in CSF, IgG and inulin were co-administered into the lateral ventricle via a catheter, and CSF was collected from the cisterna magna via another catheter time-sequentially. Blood was also obtained from the same individuals, and the concentrations of IgG and inulin in CSF and plasma were measured. The results revealed that PK parameters of IgG were similar to those of inulin; half-life and clearance of IgG were 47.0 ± 6.49 min and 29.0 ± 15.2 mL/day/kg, and those of inulin were 52.8 ± 25.4 min and 29.0 ± 13.3 mL/day/kg. Moreover, deconvolution analysis indicated that all of the IgG administered in the lateral ventricle was transferred to plasma from CSF within 24 hours. This study demonstrated that IgG in CSF was eliminated by bulk flow and transferred totally to blood circulation.     

Antinociceptive cross-tolerance between [D-Arg2]-dermorphin tetrapeptide analogs and morphine

Cross-tolerance between [D-Arg2]-dermorphin tetrapeptide analogs and morphine with respect to antinociception was examined in the present set of experiments. Systemic administration of H-Tyr-D-Arg-Phe-Gly-NH2 (TDAPG-NH2), H-Tyr-D-Arg-Phe-beta-Ala-OH (TDAPA) or morphine over a period of 5 days produced the development of tolerance. In the cross-tolerance study, antinociception after subcutaneous (SC), intracerebroventricular (ICV) and intrathecal (IT) administrations of TDAPG-NH2 and TDAPA in morphine-tolerant mice was not significantly different from their respective effects in saline-pretreated control mice. A marked tolerance to SC- and ICV-administered morphine was seen in mice made tolerant to TDAPG-NH2 and TDAPA. However, IT administration of morphine produced no significant decrement in the antinociceptive activity in mice made tolerant to TDAPG-NH2 and TDAPA. These data indicate that [D-Arg2]-dermorphin tetrapeptide analogs can produce significant antinociception in morphine-tolerant mice.     

Role of KATP channels in reduced antinociceptive effect of morphine in streptozotocin-induced diabetic mice

The nociceptive effect was measured using withdrawal latency in tail flick test in mice rendered diabetic by administering streptozotocin (200 mg/kg, i.p.). The antinociceptive effect of morphine (4 and 8 mg/kg, s.c.) and cromakalim, a KATP channel opener, (0.3, 1 and 2 micrograms, i.c.v.) was significantly reduced in diabetic mice. Moreover, co-administration of cromakalim(0.3 microgram) did not alter the reduced antinociceptive effect of morphine(4 mg/kg) in diabetic mice. Spleenectomy in diabetic mice restored the decrease in antinociceptive effect of morphine and cromakalim. Multiple dose treatment with insulin to maintain euglycaemia for 3 days in diabetic mice prevented the decrease in antinociceptive effect of morphine and cromakalim. However, hyperglycaemic tyrode's buffer did not alter the pD2 value of morphine in isolated guinea pig ileum suggesting that hyperglycaemia does not interfere with mu receptor mediated responses in vitro. The results suggest that hyperglycaemia induced decrease in antinociceptive effect of morphine and cromakalim may be due to alteration in KATP channels. Some unknown factor from spleen in diabetic mice may be responsible for this alteration in KATP channels in diabetic mice.     

Diuretic and natriuretic action of adrenomedullin administered intracerebroventricularly in conscious rats

Intracerebroventricular administration of rat adrenomedullin (AM) to conscious hydrated or salt-loaded rats, resulted in a significant increase in urinary volume. The diuretic effect of adrenomedullin occurred during the 6-h period of urine collection and was most effective during the 3 and 6 h. Most remarkably, AM given IVT induced a dose-related increase in urinary sodium excretion at all periods of urine collection. In addition, AM induced significant kaliuresis. Our results strongly suggest that AM may play a significant role in the central regulation of fluid and electrolyte homeostasis, and that its diuretic and natriuretic effect may be, at least in part, centrally mediated.     

Involvement of p38 Mapkinase in attenuation of antinociceptive effect of morphine in diabetic mice

Experimental diabetes induced by streptozotocin (200 mg/kg, ip) markedly decreased the antinociceptive effect of morphine and significantly increased the urinary nitrite concentration. Administration of FR-167653 (a selective p38MAPKinase inhibitor) in a dose of 4 mg/kg improved the antinociceptive effect of morphine and attenuated the increase in urinary nitrite concentration in diabetic mice. It may be concluded that diabetes-induced decrease in antinociceptive effect of morphine may be due to induction of p38 MAPKinase activity.     

Differential regulation of morphine antinociceptive effects by endogenous enkephalinergic system in the forebrain of mice

Background

Mice lacking the preproenkephalin (ppENK) gene are hyperalgesic and show more anxiety and aggression than wild-type (WT) mice. The marked behavioral changes in ppENK knock-out (KO) mice appeared to occur in supraspinal response to painful stimuli. However the functional role of enkephalins in the supraspinal nociceptive processing and their underlying mechanism is not clear. The aim of present study was to compare supraspinal nociceptive and morphine antinociceptive responses between WT and ppENK KO mice.

Results

The genotypes of bred KO mice were confirmed by PCR. Met-enkephalin immunoreactive neurons were labeled in the caudate-putamen, intermediated part of lateral septum, lateral globus pallidus, intermediated part of lateral septum, hypothalamus, and amygdala of WT mice. Met-enkephalin immunoreactive neurons were not found in the same brain areas in KO mice. Tail withdrawal and von Frey test results did not differ between WT and KO mice. KO mice had shorter latency to start paw licking than WT mice in the hot plate test. The maximal percent effect of morphine treatments (5 mg/kg and 10 mg/kg, i.p.) differed between WT and KO mice in hot plate test. The current source density (CSD) profiles evoked by peripheral noxious stimuli in the primary somatosenstory cortex (S1) and anterior cingulate cortex (ACC) were similar in WT and KO mice. After morphine injection, the amplitude of the laser-evoked sink currents was decreased in S1 while the amplitude of electrical-evoked sink currents was increased in the ACC. These differential morphine effects in S1 and ACC were enhanced in KO mice. Facilitation of synaptic currents in the ACC is mediated by GABA inhibitory interneurons in the local circuitry. Percent increases in opioid receptor binding in S1 and ACC were 5.1% and 5.8%, respectively.

Conclusion

The present results indicate that the endogenous enkephalin system is not involved in acute nociceptive transmission in the spinal cord, S1, and ACC. However, morphine preferentially suppressed supraspinal related nociceptive behavior in KO mice. This effect was reflected in the potentiated differential effects of morphine in the S1 and ACC in KO mice. This potentiation may be due to an up-regulation of opioid receptors. Thus these findings strongly suggest an antagonistic interaction between the endogenous enkephalinergic system and exogenous opioid analgesic actions in the supraspinal brain structures.     

Biosynthesis of beta-endorphin by the neurointermediate lobes from rats treated with morphine or alcohol

Rats were rendered tolerant to either morphine or alcohol, by 21- day drug treatment. The neurointermediate lobes (NIL) were removed and incubated with [³H]-phenylalanine for 3 hrs. The biosynthesized pro-opiomelanocortin (POMC), β-lipotropin (β-LPH) and β-endorphin- like peptides (β-EPLPs) were purified from the total protein extract of the NIL by immunoprecipitation with an antiserum to β-endorphin (β-EP), and analyzed by sodium dodecyl sulfate polyacrylamide disc gel elecrophoresis. The β-EPLPs were further characterized by extraction from the gel and microsequencing. The homology of rat POMC to authentic bovine POMC was established by extraction from the gel and peptide mapping of its tryptic digestion products. Furthermore, the β-endorphin like immunoreactivity (β-EPLI) was estimated in the incubation medium and in the NIL extract. The morphine treatment induced a decrease in the degree of incorporation of [³H]- phenylalanine into POMC, β-LPH and β-EPLPs, associated with a decrease in the content of β-EPLI in the NIL extract and in the incubation medium. Alcohol induced an increase in the degree of incorporation of [³H]-phenylalanine into POMC, β-LPH and β-EPLPs, and an increase in the β-EPLI content in the incubation medium, but no change in the β-EPLI in the NIL extract. These results indicate an effect of chronic morphine and alcohol treatment on the biosynthesis and release of β-EPLPs by the NIL.     

Differential potentiative effects of GABA receptor agonists in the production of antinociception induced by morphine and beta-endorphin administered intrathecally in the mouse

The effect of muscimol or baclofen injected intrathecally (i.t.) on the inhibition of the tail-flick response induced by morphine and beta-endorphin administered i.t. was studied in ICR mice. The i.t. injection of muscimol (100 ng) or baclofen (10 ng) alone did not affect the basal inhibition of the tail-flick response. Morphine (0.2 microg) and beta-endorphin (0.1 microg) caused only slight inhibition of the tail-flick response. Baclofen, but not muscimol, injected i.t. enhanced the inhibition of the tail-flick response induced by i.t. administered morphine. Both muscimol and baclofen injected i.t. significantly enhanced i.t. injected beta-endorphin-induced inhibition of the tail-flick response. Our results suggest that the GABA(B), but not GABA(A), receptors located in the spinal cord appear to be involved in enhancing the inhibition of the tail-flick response induced by morphine administered spinally. In addition, both GABA(A) and GABA(B) receptors are involved in enhancing the inhibition of the tail-flick response induced by beta-endorphin administered i.t.