Techniques for assessing knee joint pain in arthritis

Background

In general, opioids that induce the recycling of μ-opioid receptors (MORs) promote little desensitization, although morphine is one exception to this rule. While morphine fails to provoke significant internalization of MORs in cultured cells, it does stimulate profound desensitization. In contrast, morphine does promote some internalization of MORs in neurons although this does not prevent this opioid from inducing strong antinociceptive tolerance.

Results

In neurons, morphine stimulates the long-lasting transfer of MOR-activated Gα subunits to proteins of the RGS-R7 and RGS-Rz subfamilies. We investigated the influence of this regulatory process on the capacity of morphine to promote desensitization and its association with MOR recycling in the mature nervous system. In parallel, we also studied the effects of [D-Ala², N-MePhe⁴, Gly-ol⁵] encephalin (DAMGO), a potent inducer of MOR internalization that promotes little tolerance. We observed that the initial exposure to icv morphine caused no significant internalization of MORs but rather, a fraction of the Gα subunits was stably transferred to RGS proteins in a time-dependent manner. As a result, the antinociception produced by a second dose of morphine administered 6 h after the first was weaker. However, this opioid now stimulated the phosphorylation, internalization and recycling of MORs, and further exposure to morphine promoted little tolerance to this moderate antinociception. In contrast, the initial dose of DAMGO stimulated intense phosphorylation and internalization of the MORs associated with a transient transfer of Gα subunits to the RGS proteins, recovering MOR control shortly after the effects of the opioid had ceased. Accordingly, the recycled MORs re-established their association with G proteins and the neurons were rapidly resensitized to DAMGO.

Conclusion

In the nervous system, morphine induces a strong desensitization before promoting the phosphorylation and recycling of MORs. The long-term sequestering of morphine-activated Gα subunits by certain RGS proteins reduces the responses to this opioid in neurons. This phenomenon probably increases free Gβγ dimers in the receptor environment and leads to GRK phosphorylation and internalization of the MORs. Although, the internalization of the MORs permits the transfer of opioid-activated Gα subunits to the RGSZ2 proteins, it interferes with the stabilization of this regulatory process and recycled MORs recover the control on these Gα subunits and opioid tolerance develops slowly.     

Lack of tolerance and morphine-induced cross-tolerance to the analgesia of chimeric peptide of Met-enkephalin and FMRFa

Chimeric peptide of Met-enkephalin and FMRFa (YGGFMKKKFMRFa-YFa), a κ-opioid receptor specific peptide, did not induce tolerance and cross-tolerance effects to its analgesic action on day 5 after pretreatment with either YFa or morphine for 4 days. However, pretreatment with YFa for 4 days led to the development of cross-tolerance to the analgesic effects of morphine and also 4 days of pretreatment of morphine resulted in the expression of tolerance to its own analgesic effects. Similar expression of tolerance and cross-tolerance were also observed when YFa was compared with the κ receptor agonist peptide dynorphin A(1–13) [DynA(1–13)]. Cross-tolerance effects between YFa and DynA(1–13) analgesia were also not observed on day 5. Interestingly, when YFa and DynA(1–13) were tested for their analgesic effects for 5 days, reduction in analgesia on day 3 was observed in case of DynA(1–13) whereas YFa maintained its analgesia for 5 days. Thus, chimeric peptide YFa may serve as a useful probe to understand pain modulation and expression of tolerance and cross-tolerance behavior with other opioids.     

Phosphoproteomics and Bioinformatics Analyses of Spinal Cord Proteins in Rats with Morphine Tolerance

Introduction

Morphine is the most effective pain-relieving drug, but it can cause unwanted side effects. Direct neuraxial administration of morphine to spinal cord not only can provide effective, reliable pain relief but also can prevent the development of supraspinal side effects. However, repeated neuraxial administration of morphine may still lead to morphine tolerance.

Methods

To better understand the mechanism that causes morphine tolerance, we induced tolerance in rats at the spinal cord level by giving them twice-daily injections of morphine (20 µg/10 µL) for 4 days. We confirmed tolerance by measuring paw withdrawal latencies and maximal possible analgesic effect of morphine on day 5. We then carried out phosphoproteomic analysis to investigate the global phosphorylation of spinal proteins associated with morphine tolerance. Finally, pull-down assays were used to identify phosphorylated types and sites of 14-3-3 proteins, and bioinformatics was applied to predict biological networks impacted by the morphine-regulated proteins.

Results

Our proteomics data showed that repeated morphine treatment altered phosphorylation of 10 proteins in the spinal cord. Pull-down assays identified 2 serine/threonine phosphorylated sites in 14-3-3 proteins. Bioinformatics further revealed that morphine impacted on cytoskeletal reorganization, neuroplasticity, protein folding and modulation, signal transduction and biomolecular metabolism.

Conclusions

Repeated morphine administration may affect multiple biological networks by altering protein phosphorylation. These data may provide insight into the mechanism that underlies the development of morphine tolerance.     

Facilitation of μ-opioid receptor activity by preventing δ-opioid receptor-mediated codegradation

δ-opioid receptors (DORs) form heteromers with μ-opioid receptors (MORs) and negatively regulate MOR-mediated spinal analgesia. However, the underlying mechanism remains largely unclear. The present study shows that the activity of MORs can be enhanced by preventing MORs from DOR-mediated codegradation. Treatment with DOR-specific agonists led to endocytosis of both DORs and MORs. These receptors were further processed for ubiquitination and lysosomal degradation, resulting in a reduction of surface MORs. Such effects were attenuated by treatment with an interfering peptide containing the first transmembrane domain of MOR?(MOR(TM1)), which interacted with DORs and disrupted the MOR/DOR interaction. Furthermore, the systemically applied fusion protein consisting of MOR(TM1) and TAT at the C terminus could disrupt the MOR/DOR interaction in the mouse spinal cord, enhance the morphine analgesia, and reduce the antinociceptive tolerance to morphine. Thus, dissociation of MORs from DORs in the cell membrane is?a potential strategy to improve opioid analgesic therapies.     

Difference in the development of tolerance to morphine and d-ala2-methionine-enkephalin in C57 BL/6J and DBA/2J mice

The analgesic effect elicited by intracerebroventricular (icv) administration of either morphine or d-ala²-methionine-enkephalin (d-ala²-met-enk) was studied during the onset and offset of morphine tolerance in DBA/2_J (DBA) and C₅₇ BL/6_J (C₅₇) strains of mice. DBA mice become tolerant to the analgesic effect of morphine icv injected after receiving 8 subcutaneous (sc) injections (2 injections daily × 4 days) of the ED₅₀ of morphine for analgesia. In c₅₇ mice tolerance to morphine icv-administered is evident after only a single sc injection of morphine ED₅₀. On the contrary the development of cross-tolerance to the analgesic effect of d-ala²-met-enk is similar in both strains of mice. With respect to the offset period, the recovery of the analgesic effect of morphine and d-ala²-met-enk is slower in C₅₇ than in DBA mice; in C₅₇ mice tolerance to both morphine and d-ala²-met-enk is still present 10 days after morphine withdrawal. These results suggest the existence of a strain dependent rate in the onset of tolerance to the analgesic effect of morphine. C₅₇ mice represent an interesting tool to investigate tolerance to opiates and opioid peptides.     

Cholera toxin and pertussis toxin on opioid- and alpha 2-mediated supraspinal analgesia in mice.

Cholera toxin, an agent that impairs the function of Gs transducer proteins, was injected (0.5 microgram/mouse, icv) and the antinociceptive activity of opioids and clonidine was studied 24h later in the tail-flick test. In these animals, an enhancement of the analgesic potency of morphine, beta-endorphin and clonidine could be observed. Cholera toxin did not modify the antinociception evoked by the enkephalin derivatives DAGO and DADLE. Pertussis toxin that catalyses the ADP ribosylation of alpha subunits of Gi/Go regulatory proteins was given icv (0.5 microgram/mouse). This treatment reduced the analgesic effect of opioids and clonidine. However, while the analgesia elicited by DAGO, DADLE and clonidine was greatly decreased, the effect of morphine and beta-endorphin was reduced to a moderate extent. It is concluded that Gi/Go regulatory proteins functionally coupled to opioid and alpha 2 receptors are implicated in the efficacy displayed by opioids and clonidine to produce supraspinal analgesia. Moreover, these two receptors are susceptible to regulation by a process that might involve a Gs protein.     

Tolerance and morphine-induced cross-tolerance are not shown to Tyr-W-MIF-1 analgesia.

Tolerance and cross-tolerance between Tyr-W-MIF-1, a mixed micro-agonist/antagonist, and morphine were examined. Opiate dependence also was examined. Rats were pretreated with Tyr-W-MIF-1, morphine, or saline for 4 days. On day 5, the animals were tested for Tyr-W-MIF-1 analgesia, morphine analgesia, or naloxone-precipitated withdrawal. Tyr-W-MIF-1- and morphine-pretreated animals showed similar levels of dependence. Animals pretreated with Tyr-W-MIF-1 failed to express tolerance to Tyr-W-MIF-1 analgesia but did display cross-tolerance to morphine analgesia. Animals pretreated with morphine displayed tolerance to morphine analgesia but did not express cross-tolerance to Tyr-W-MIF-1 analgesia. Therefore, tolerance and morphine-induced cross-tolerance were not expressed to Tyr-W-MIF-1 analgesia.     

Retention of supraspinal delta-like analgesia and loss of morphine tolerance in delta opioid receptor knockout mice

Gene targeting was used to delete exon 2 of mouse DOR-1, which encodes the delta opioid receptor. Essentially all 3H-[D-Pen2,D-Pen5]enkephalin (3H-DPDPE) and 3H-[D-Ala2,D-Glu4]deltorphin (3H-deltorphin-2) binding is absent from mutant mice, demonstrating that DOR-1 encodes both delta1 and delta2 receptor subtypes. Homozygous mutant mice display markedly reduced spinal delta analgesia, but peptide delta agonists retain supraspinal analgesic potency that is only partially antagonized by naltrindole. Retained DPDPE analgesia is also demonstrated upon formalin testing, while the nonpeptide delta agonist BW373U69 exhibits enhanced activity in DOR-1 mutant mice. Together, these findings suggest the existence of a second delta-like analgesic system. Finally, DOR-1 mutant mice do not develop analgesic tolerance to morphine, genetically demonstrating a central role for DOR-1 in this process.     

Mu-Opioid Receptors Transiently Activate the Akt-nNOS Pathway to Produce Sustained Potentiation of PKC-Mediated NMDAR-CaMKII Signaling

Background

In periaqueductal grey (PAG) matter, cross-talk between the Mu-opioid receptor (MOR) and the glutamate N-methyl-D-Aspartate receptor (NMDAR)-CaMKII pathway supports the development of analgesic tolerance to morphine. In neurons, histidine triad nucleotide binding protein 1 (HINT1) connects the regulators of G protein signaling RGSZ1 and RGSZ2 to the C terminus of the MOR. In response to morphine, this HINT1-RGSZ complex binds PKCγ, and afterwards, the interplay between PKCγ, Src and Gz/Gi proteins leads to sustained potentiation of NMDAR-mediated glutamate responses.

Methodology/Principal Findings

Following an intracerebroventricular (icv) injection of 10 nmol morphine, Akt was recruited to the synaptosomal membrane and activated by Thr308 and Ser473 phosphorylation. The Akt activation was immediately transferred to neural Nitric Oxide Synthase (nNOS) Ser1417. Afterwards, nitric oxide (NO)-released zinc ions recruited PKCγ to the MOR to promote the Src-mediated phosphorylation of the Tyr1325 NMDAR2A subunit. This action increased NMDAR calcium flux and CaMKII was activated in a calcium-calmodulin dependent manner. CaMKII then acted on nNOS Ser847 to produce a sustained reduction in NO levels. The activation of the Akt-nNOS pathway was also reduced by the binding of these proteins to the MOR-HINT1 complex where they remained inactive. Tolerance to acute morphine developed as a result of phosphorylation of MOR cytosolic residues, uncoupling from the regulated G proteins which are transferred to RGSZ2 proteins. The diminished effect of morphine was prevented by LNNA, an inhibitor of nNOS function, and naltrindole, a delta-opioid receptor antagonist that also inhibits Akt.

Conclusions/Significance

Analysis of the regulatory phosphorylation of the proteins included in the study indicated that morphine produces a transient activation of the Akt/PKB-nNOS pathway. This activation occurs upstream of PKCγ and Src mediated potentiation of NMDAR activity, ultimately leading to morphine tolerance. In summary, the Akt-nNOS pathway acts as a primer for morphine-triggered events which leads to the sustained potentiation of the NMDAR-CaMKII pathway and MOR inhibition.     

Activation of mu-opioid receptors transfers control of Galpha subunits to the regulator of G-protein signaling RGS9-2: role in receptor desensitization

In mouse periaqueductal gray matter (PAG) membranes, the mu-opioid receptor (MOR) coprecipitated the alpha-subunits of the Gi/o/z/q/11 proteins, the Gbeta1/2 subunits, and the regulator of G-protein signaling RGS9-2 and its partner protein Gbeta5. RGS7 and RGS11 present in this neural structure showed no association with MOR. In vivo intracerebroventricular injection of morphine did not alter MOR immunoreactivity, but 30 min and 3 h after administration, the coprecipitation of Galpha subunits with MORs was reduced by up to 50%. Furthermore, the association between Galpha subunits and RGS9-2 proteins was increased. Twenty-four hours after receiving intracerebroventricular morphine, the Galpha subunits left the RGS9-2 proteins and re-associated with the MORs. However, doses of the opioid able to induce tolerance promoted the stable transfer of Galpha subunits to the RGS9-2 control. This was accompanied by Ser phosphorylation of RGS9-2 proteins, which increased their co-precipitation with 14-3-3 proteins. In the PAG membranes of morphine-desensitized mice, the capacity of the opioid to stimulate G-protein-related guanosine 5'-O-(3-[35S]thiotriphosphate) binding as well as low Km GTPase activity was attenuated. The in vivo knockdown of RGS9-2 expression prevented morphine from altering the association between MORs and G-proteins, and tolerance did not develop. In PAG membranes from RGS9-2 knockdown mice, morphine showed full capacity to activate G-proteins. Thus, the tolerance that develops following an adequate dose of morphine is caused by the stabilization and retention of MOR-activated Galpha subunits by RGS9-2 proteins. This multistep process is initiated by the morphine-induced transfer of MOR-associated Galpha subunits to the RGS9-2 proteins, followed by Ser phosphorylation of the latter and their binding to 14-3-3 proteins. This regulatory mechanism probably precedes the loss of MORs from the cell membrane, which has been observed with other opioid agonists.     

Effect of methamphetamine on the development of acute morphine tolerance and dependence in mice

The effect of methamphetamine on morphine analgesia (tail-flick assay) was studied in non-tolerant mice and in mice made acutely tolerant to morphine following a single injection of 100 mg/kg morphine. The analgesic potency of morphine was increased in non-tolerant and tolerant mice to the same extent by 3.2 mg/kg methamphetamine (3.3 and 4.4 fold increases, respectively). In contrast, the ED50's for morphine analgesia and naloxone-precipitated jumping in mice pretreated with either 100 mg/kg morphine or both morphine and 3.2 mg/kg methamphetamine were not significantly different, indicating that methamphetamine had no effect on the development of acute morphine tolerance and dependence. Although methamphetamine had no effect on the development of acute tolerance to morphine, 4-day pretreatment with methamphetamine produced cross-tolerance to morphine analgesia. However, cross-tolerance to morphine was not accompanied by enchanced sensitivity to naloxone.     

Further characterization of alpha N-acetyl beta-endorphin-(1-31) regulatory activity, I: Effect on opioid- and alpha 2-mediated supraspinal antinociception in mice.

Picomol doses of the acetylated derivative of beta-endorphin-(1-31), injected intracerebroventricularly (icv) in mice, reduced the analgesic activity of morphine, etorphine and beta-endorphin-(1-31), while the efficiency of DAGO and DADLE in producing analgesia was enhanced. The effects of the delta agonists DPDPE and [D-Ala2]-Deltorphin II were not altered by this treatment. After alpha N-acetyl beta-endorphin-(1-31) injection, morphine antagonized the analgesia of DAGO. The regulatory effect of alpha N-acetyl beta-endorphin-(1-31) was exhibited when giving the peptide both before (up to 24 h) and after the opioids. Naloxone did not prevent or reverse that modulatory activity; moreover, pretreatment with the acetylated peptide did not change the pA2 value displayed by the antagonist at the mu receptor. The antinociceptive activity of the alpha 2-adrenoceptor agonist clonidine was also increased in mice treated with alpha N-acetyl beta-endorphin-(1-31). The reducing activity of alpha N-acetyl beta-endorphin-(1-31) upon morphine- and beta-endorphin-induced analgesia was not exhibited in mice undergoing treatment with pertussis toxin or N-ethylmaleimide, agents known to impair the function of Gi/Go transducer proteins. However, the enhancing activity displayed by this peptide upon DAGO- DADLE and clonidine-evoked antinociception was still manifested. These results confirm and strengthen the idea of alpha N-acetyl beta-endorphin-(1-31) acting as a non-competitive regulator of mu opioid- and alpha 2-adrenoceptor-mediated supraspinal antinociception. A neural substrate acted on by both receptors (likely Gi/Go transducer proteins) appears to be involved in the effects of that neuropeptide.     

Tolerance to effects of clonidine and morphine on sulfobromophthalein disposition in mice

Chronic treatment of mice with clonidine or morphine caused tolerance to the analgesic and thermoregulatory effects of these drugs. After chronic morphine, mice also became tolerant to the analgesic and thermoregulatory effects of clonidine. Cross tolerance to the hypothermic effect of morphine was demonstrated after chronic clonidine administration, but no diminution of morphine-induced analgesia could be shown. Morphine and clonidine acutely increased the retention of sulfobromophthalein (BSP) in plasma and liver. Chronic dosing with morphine or clonidine caused partial tolerance and cross-tolerance to the rise in hepatic BSP caused by an acute challenge with either agonist. However, both drugs elevated plasma BSP levels similarly in tolerant and non-tolerant mice. Thus, regimens which readily induced tolerance to the analgesic and hypothermic effects of morphine or clonidine were only partially effective in modifying the acute hepatobiliary effects of these drugs.     

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.     

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.     

Acute cross-tolerance to opioids in heroin delta-opioid-responding Swiss Webster mice

It is generally thought that the mu receptor actions of metabolites, 6-monoacetylmorphine (6MAM) and morphine, account for the pharmacological actions of heroin. However, upon intracerebroventricular (i.c.v.) administration in Swiss Webster mice, heroin and 6MAM act on delta receptors while morphine acts on mu receptors. Swiss Webster mice made tolerant to subcutaneous (s.c.) morphine by morphine pellet were not cross-tolerant to s.c. heroin (at 20 min in the tail flick test). Now, opioids were given in combination, s.c. (6.5 h) and i.c.v. (3 h) preceding testing the challenging agonist i.c.v. (at 10 min in the tail flick test). The combination (s.c. + i.c.v.) morphine pretreatment induced tolerance to the mu action of morphine but no cross-tolerance to the delta action of heroin, 6MAM and DPDPE and explained why morphine pelleting did not produce cross-tolerance to s.c. heroin above. Heroin plus heroin produced tolerance to delta agonists but not to mu agonists. Surprisingly, all combinations of morphine with the delta agonists produced tolerance to morphine which now acted through delta receptors (inhibited by i.c.v. naltrindole), an unusual change in receptor selectivity for morphine.     

THIP analgesia: cross tolerance with morphine

THIP (4,5,6,7-tetrahydroisoxazolo (5,4-c) pyridone-3-ol), a direct acting GABA receptor agonist, has been shown to have antinociceptive properties. To determine whether tolerance develops to the analgesic response, mice received multiple injections of THIP for up to 21 days after which analgesia was tested using both tail immersion and hot-plate methods. Both tests indicated a significant reduction in the antinociceptive response to THIP, as well as other GABA agonists, beginning between days 3 and 5 of chronic administration. Moreover, these animals demonstrated a decreased analgesic response to morphine, and morphine tolerant animals were also less responsive to THIP. These data indicate that opiates and GABA agonists induce analgesia by acting through separate but related pathways in the central nervous system.     

NMDAR-nNOS generated zinc recruits PKCgamma to the HINT1-RGS17 complex bound to the C terminus of Mu-opioid receptors

In neurons, the C terminus of the Mu-opioid receptor (MOR) binds to the protein kinase C-interacting protein/histidine triad nucleotide binding protein 1 (PKCI/HINT1) which in turn binds the regulator of G-protein signalling RGSZ1/Z2 (RGSZ) protein. In this study, we found that intracerebroventricular (icv) administration of morphine recruits PKC isoforms, mostly PKCgamma, to the MOR via the HINT1/RGSZ complex. There, diacylglycerol (DAG) activates this PKCgamma to phosphorylate the MOR and thus, its signal strength was reduced. When PKCI/HINT1 expression is depressed, morphine produces stronger analgesic effects and neither the PKCgamma-MOR complex nor serine phosphorylation of this receptor is detected. This MOR-PKC association involves the cysteine rich domains (CRDs) in the regulatory C1 region of PKC, as well as requiring free zinc ions, HINT1 and RGSZ proteins. Increasing the availability of this metal ion recruits inactive PKCgamma to the MOR, while phorbol esters prevent this binding and even disrupt it. The nitric oxide donor (S)-Nitroso-N-acetylpenicillamine (SNAP) foments the association of PKCgamma with the MORs, effect that was prevented by the heavy metal chelator N,N,N',N'-tetrakis(2-pyridylmethyl) ethylenediamine (TPEN), suggesting a role for endogenous zinc and neural nitric oxide synthase. The N-methyl-D-aspartate receptor (NMDAR) antagonist, MK801, also prevented PKCgamma recruitment to MORs and serine phosphorylation of the receptors following icv morphine. These results indicate that the NMDAR/nNOS cascade, activated via MORs, provide the free zinc ions required for inactive PKCgamma to bind to HINT1/RGSZ complex at the C terminus of the receptor.     

Possible participation of endogenous opioid peptides on the mechanism involved in analgesia induced by vouacapan.

The involvement of opioid peptides in the mechanism of action of vouacapan, a new experimental compound extracted from seeds of Pterodon poligalaeflorus Benth, was investigated both in mice utilizing acetic acid writhing response and in rats utilizing inflammatory hyperalgesia induced by carrageenan and modified Randall-Selitto method. Vouacapan, in both models, caused a dose-dependent analgesia when injected p.o., s.c. and i.p. The analgesic effect was partially blocked by naloxone, nalorphine and n-methyl-nalorphine. Significant tolerance to analgesic effect was observed following repeated administration of vouacapan or morphine. On the last day of treatment, cross administration revealed symmetrical and asymmetrical cross-tolerance between vouacapan and morphine, in rats and mice, respectively. We conclude that a release of endorphins could be involved in the analgesic mechanism of vouacapan in both models tudied.     

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.