CXBK Mice Research Articles

Involvement of spinal release of α-neo-endorphin on the antinociceptive effect of TAPA

The antinociceptive effect of i.t.-administered Tyr-d-Arg-Phe-β-Ala (TAPA), an N-terminal tetrapeptide analog of dermorphin, was characterized in ddY mice. In the mouse tail-flick test, TAPA administered i.t. produced a potent antinociception. The antinociception induced by TAPA was significantly attenuated by i.t. pretreatment with the κ-opioid receptor antagonist nor-binaltorphimine, as well as by the μ-opioid receptor antagonist β-funaltrexamine and the μ1-opioid receptor antagonist naloxonazine. TAPA-induced antinociception was also significantly suppressed by co-administration of the μ1-opioid receptor antagonist Tyr-d-Pro-Phe-Phe-NH2 (d-Pro2-endomorphin-2) but not by co-administration of the μ2-opioid receptor antagonists Tyr-d-Pro-Trp-Phe-NH2 (d-Pro2-endomorphin-1) and Tyr-d-Pro-Trp-Gly-NH2 (d-Pro2-Tyr-W-MIF-1). In CXBK mice whose μ1-opioid receptors were naturally reduced, the antinociceptive effect of TAPA was markedly suppressed compared to the parental strain C57BL/6ByJ mice. Moreover, the antinociception induced by TAPA was significantly attenuated by i.t. pretreatment with antiserum against the endogenous κ-opioid peptide α-neo-endorphin but not antisera against other endogenous opioid peptides. In prodynorphin-deficient mice, the antinociceptive effect of TAPA was significantly reduced compared to wild-type mice. These results suggest that the spinal antinociception induced by TAPA is mediated in part through the release of α-neo-endorphin in the spinal cord via activation of spinal μ1-opioid receptors.

Ethanol drinking-in-the-dark facilitates behavioral sensitization to ethanol in C57BL/6J, BALB/cByJ, but not in mu-opioid receptor deficient CXBK mice

BackgroundNeuroplasticity associated with drug-induced behavioral sensitization has been associated with excessive drug pursuit and consumption characteristic of addiction. Repeated intraperitoneal (ip) injections of ethanol (EtOH) can induce psychomotor sensitization in mice. In terms of its clinical relevance, however, it is important to determine whether this phenomenon can also be produced by voluntary EtOH consumption. MethodsThe present investigation used a drinking-in-the-dark (DID) methodology to induce high levels of EtOH drinking in mice; EtOH replaces water for 2 or 4h, starting 3h after the beginning of the dark cycle. Animals followed a 3-week DID protocol prior to an evaluation of EtOH-induced locomotor activity (acute and repeated EtOH). For the first week, animals had access to 20% EtOH. On weeks 2 and 3, different concentrations of EtOH (10, 20 or 30%) were used. Three different inbred strains of mice were used: C57BL/6J (B6), BALB/cByJ (BALB), and CXBK. The CXBK mouse line was used because of its reduced expression and functioning of brain mu-opioid receptors, which have been suggested to participate in the development of EtOH-induced sensitization. B6 and BALB mice were used as controls. ResultsB6 and CXBK mice presented comparable levels of EtOH drinking (approx. 3g/kg in 2h), that were higher than those showed by BALB. All animals, regardless of genotype, adjusted volume of EtOH intake to obtain stable g/kg of EtOH across concentrations. Previous EtOH DID produced (B6) or potentiated (BALB) sensitization to EtOH; this effect was not seen in CXBK. Western blot analysis showed a reduced number of mu-opioid receptors in several brain regions of CXBK as compared to that of B6 and BALB mice. ConclusionsIn summary, here we show that the DID methodology can be used to trigger EtOH-induced neuroplasticity supporting psychomotor sensitization, a process that might require participation of mu-opioid receptors.

Is there genetic polymorphism evidence for individual human sensitivity to opiates?

Opiate analgesics have been widely used for severe acute pain and chronic cancer-related pain. Individual differences in the effectiveness of opiates and their side effects limit the clinical benefits and increase risks of drug abuse. Genetic factors might affect variations of opiate sensitivity. The mu opioid peptide receptor (MOP) is the principal site of pharmacologic actions for most clinically important opiate drugs. Recent studies using various knockout mice and recombinant-inbred strain CXBK mice have indicated that the analgesic effect of morphine is dependent on the amount of the MOP. There are more than 100 polymorphisms identified in the human MOP (OPRM1) gene. These polymorphisms might be correlated with OPRM1 mRNA stability and opiate sensitivity, including opiate analgesia, tolerance, and dependence. More precise studies on the relationship between gene polymorphisms and opiate sensitivity will enable realization of personalized pain treatment by predicting opiate sensitivity and requirement for each patient.

Intracisternal A-particle element in the 3′ noncoding region of the mu-opioid receptor gene in CXBK mice: a new genetic mechanism underlying differences in opioid sensitivity

CXBK mice, recombinant inbred mice derived from C57BL/6By and BALB/cBy progenitors, display reduced morphine-induced analgesia. Earlier we reported that CXBK mice expressed a reduced amount of the major transcript, MOR-1 mRNA, of the mu-opioid receptor gene. The CXBK MOR-1 mRNA contains a normal coding region and an abnormally long untranslated region. To identify the nucleotide-sequence difference between the CXBK MOR-1 mRNA and that of the progenitors, we first characterized the 3' untranslated region of the MOR-1 mRNA, which was largely unknown. A 3' rapid amplification of cDNA ends-PCR analysis revealed that the 3' untranslated region of the C57BL/6By MOR-1 mRNA was 10 181 nucleotides transcribed from an exon. Next, we compared the MOR-1 genes in C57BL/6By, CXBK, and BALB/cBy mice, and found a 5293 nucleotide insertion only in CXBK mice. The inserted sequence was a variant of the intracisternal A-particle elements that exist in the mouse genome at approximately 1000 sites. Reverse transcription-PCR analyses revealed that the intracisternal A-particle element was transcribed as a part of the CXBK MOR-1 mRNA. No other differences were found in the MOR-1 mRNA between CXBK and BALB/cBy mice, whereas 100 nucleotides differed between C57BL/6By and CXBK mice aside from the intracisternal A-particle insertion. Finally, CXBK mice displayed reduced morphine responses compared with BALB/cBy mice. Our data suggest that differences in the MOR-1 3' untranslated region appear to cause the CXBK phenotype. This genetic mechanism underlying the CXBK phenotype may provide good insight into the possible genetic mechanisms underlying individual differences in opioid sensitivity in humans.

Involvement of the 3′ non‐coding region of the mu opioid receptor gene in morphine‐induced analgesia

Abstract The mu opioid receptor (MOR) is known to play an essential role in morphine‐induced analgesia. MOR‐1 mRNA, the major MOR transcript, possesses a long 3′ untranslated region (3′UTR) in both mouse and human species. The sequence of the MOR‐1 3′UTR, especially that of its 3′ end region, is conserved between mice and humans. In the MOR‐1 3′UTR, AU‐rich elements (AREs) are densely localized at the 3′ end region, suggesting low stability of this mRNA. Numerous putative transcription factor‐binding motifs are located in the 3′ non‐coding regions of the mouse and human MOR genes. The CXBK mouse strain, known as a MOR‐deficient strain, possesses a decreased amount of MOR‐1 mRNA containing an abnormally long MOR‐1 3′UTR with a long nucleotide insertion. This insert might disrupt the stability of the MOR‐1 mRNA or might reduce the transcription of the MOR‐1 mRNA by separating the transcription factor‐binding motifs in the 3′ non‐coding region of the MOR gene, thereby decreasing MOR‐1 mRNA expression and attenuating morphine‐induced analgesia in CXBK mice. These recent findings suggest that the MOR‐1 3′UTR is involved in mRNA expression and in the difference in response to morphine. This possible genetic mechanism may provide a good starting point for designing effective pain treatments with opiates.

Differential Expression of μ-Opioid Receptor Gene in CXBK and B6 Mice by Sp1

Differential Expression of μ-Opioid Receptor Gene in CXBK and B6 Mice by Sp1

Differential Expression of -Opioid Receptor Gene in CXBK and B6 Mice by Sp1

Differential Expression of -Opioid Receptor Gene in CXBK and B6 Mice by Sp1

Reduced expression of a novel mu-opioid receptor (MOR) subtype MOR-1B in CXBK mice: implications of MOR-1B in the expression of MOR-mediated responses.

A novel mu-opioid receptor (MOR) subtype, named MOR-1B, derived from alternatively spliced variants of MOR gene, has been isolated from the rat brain. Here we found for the first time that CXBK recombinant-inbred mice display a significant reduction in the expression of MOR-1B mRNA in the brain as compared to that in their progenitor C57BL/6 mice. In contrast, the expression level of MOR-1 mRNA in the brain of CXBK mice was similar to that found in C57BL/6 mice. Furthermore, relatively lower levels of MOR-1B immunoreactivity were detected in the periaqueductal grey matter (PAG) of CXBK mice than that observed in C57BL/6 mice. To investigate further the possible changes in MOR function to activate G-proteins under the condition of a reduced MOR-1B expression, the guanosine-5'-o-(3-[35S]thio)triphosphate ([35S]GTPgammaS) binding assay was performed. We found that the increased level of [35S]GTPgammaS bindings to whole brain membranes induced by a selective MOR agonist endomorphin-1 was significantly decreased in CXBK mice, indicating that CXBK strain can be classified as MOR-1B-knockdown mice. We next investigated whether intracerebroventricular (i.c.v.) pretreatment with an antisence oligodeoxynucleotide against exon 5 of MOR gene (MOR-1B) could affect the endomorphin-1-induced supraspinal antinociception. The i.c.v. pretreatment with antisence oligodeoxynucleotide against MOR-1B produced a significant reduction in the i.c.v.-administered endomorphin-1-induced antinociceptive effect. The present data provide first evidence that a lack of MOR-1B expression may, at least in part, contribute to the reduced sensitivity to MOR agonists in CXBK mice, and MOR-1B may play a potential role in the MOR-mediated supraspinal antinociception.

Differential sensitivities of mouse strains to morphine and [Dmt 1]DALDA analgesia

[Dmt 1]DALDA (H–Dmt– d-Arg–Phe–Lys–NH 2), is a highly potent and selective μ-opioid agonist. Nevertheless, systemic [Dmt 1]DALDA retained its analgesic actions in MOR-1 knockout animals and CXBK mice despite the inactivity of morphine in these mice. [Dmt 1]DALDA was 6-fold less potent in C57BL/6J mice than in CD-1 mice, whereas morphine potency did not differ between the two strains. Thus, [Dmt 1]DALDA is a highly selective μ-opioid analgesic with significant pharmacological differences with the prototypic μ-opioid morphine.

14-Methoxymetopon, a very potent μ-opioid receptor-selective analgesic with an unusual pharmacological profile

14-Methoxymetopon is a potent opioid analgesic. When given systemically, it is approximately 500-fold more active than morphine. However, this enhanced potency is markedly increased with either spinal or supraspinal administration, where its analgesic activity is more than a million-fold greater than morphine. It was μ-opioid receptor selective in binding assays and its analgesia was blocked only by μ-opioid receptor-selective antagonists. Yet, it had a different selectivity profile than either morphine or morphine-6β-glucuronide. Unlike morphine, 14-methoxymetopon was antagonized by 3- O-methylnaltrexone, it was sensitive to antisense probes targeting exons 1, 2 and 8 of the opioid receptor gene and was inactive both spinally and supraspinally in CXBK mice. Although it retarded gastrointestinal transit, it displayed a ceiling effect with no dose lowering transit by more than 65%, in contrast to the complete inhibition of transit by morphine. These finding demonstrate that 14-methoxymetopon is a highly potent μ-opioid with a pharmacological profile distinct from that of the traditional μ-opioid morphine.

Possible involvement of μ 1-opioid receptors in the fentanyl- or morphine-induced antinociception at supraspinal and spinal sites

Fentanyl has been shown to be a potent analgesic with a lower propensity to produce tolerance and physical dependence in the clinical setting. The present study was designed to investigate the mechanisms of fentanyl- or morphine-induced antinociception at both supraspinal and spinal sites. In the mouse tail-flick test, the antinociceptive effects induced by both fentanyl and morphine were blocked by either the μ 1-opioid receptor antagonist naloxonazine or the μ 1/μ 2-opioid receptor antagonist β-funaltrexamine (β-FNA) after s.c., i.c.v. or i.t. injection. In contrast, both fentanyl and morphine given i.c.v. or i.t. failed to produce antinociception in μ 1-deficient CXBK mice. These findings indicate that like morphine, the antinociception induced by fentanyl may be mediated predominantly through μ 1-opioid receptors at both supraspinal and spinal sites in mice. We also determined the ED 50 values for s.c.-, i.c.v.- and i.t.-administered fentanyl- or morphine-induced antinociception in mice. The ED 50 values for s.c.-, i.c.v.- and i.t.-administered fentanyl-induced antinociception were 73.7, 18.5 and 1.2-fold lower than that of morphine, respectively. The present data clearly suggest the usefulness of peripheral treatment with fentanyl for the control of pain.

Lack of the involvement of μ 1-opioid receptor subtype on motivational effects induced by the endogenous μ-opioid receptor ligands endomorphin-1 and -2 in the mouse

The present study was designed to investigate the role of μ-opioid receptor subtypes in the motivational effect of endogenous μ-opioid receptor ligands, endomorphin-1 and -2. In C57BL/6J mice, endomorphin-1 produced a significant place preference, whereas endomorphin-2 exhibited a significant place aversion. These effects were abolished by a μ 1/μ 2-opioid receptor antagonist β-funaltrexamine. Under these conditions, both endomorphin-1 and -2 produced their motivational effects in μ 1-opioid receptor-deficient CXBK mice, indicating the μ 2-opioid receptor involvement. Furthermore, in the lower midbrain including ventral tegmental area, both endomorphin-1 and -2 equally produced dose-related increases in guanosine-5′- o-(3-[ 35S] thio) triphosphate bindings in C57BL/6J and CXBK mice. These findings indicate that endomorphin-1 and -2 may produce distinct motivational effects via respective μ 2-opioid receptor isoforms in the mouse. Furthermore, endomorphin-1 and -2 produced the μ 1-resistant G-protein activation in the mouse lower midbtrain.

Pharmacological characterization of the dermorphin analog [Dmt 1]DALDA, a highly potent and selective μ-opioid peptide

The dermorphin-derived peptide [Dmt 1]DALDA (H-Dmt- d-Arg-Phe-Lys-NH 2), labels μ-opioid receptors with high affinity and selectivity in receptor binding assays. In mouse, radiant heat tail-flick assay [Dmt 1]DALDA produced profound spinal and supraspinal analgesia, being approximately 5000- and 100-fold more potent than morphine on a molar basis, respectively. When administered systemically, [Dmt 1]DALDA was over 200-fold more potent than morphine. Pharmacologically, [Dmt 1]DALDA was distinct from morphine. [Dmt 1]DALDA displayed no cross-tolerance to morphine in the model used and it retained supraspinal analgesic activity in morphine-insensitive CXBK mice. Supraspinally, it also differed from morphine in its lack of sensitivity towards naloxonazine. Finally, in antisense mapping studies, [Dmt 1]DALDA was insensitive to MOR-1 exon probes that reduced morphine analgesia, implying a distinct receptor mechanism of action. Thus, [Dmt 1]DALDA is an interesting and extraordinarily potent, systemically active peptide analgesic, raising the possibility of novel approaches in the design of clinically useful drugs.

The Untranslated Region of μ-Opioid Receptor mRNA Contributes to Reduced Opioid Sensitivity in CXBK Mice

It is well known that there are individual differences in a sensitivity to analgesics. Several lines of evidence have suggested that the level of opioid-induced analgesia is dependent on the level of expression of the mu-opioid receptor (mu-OR). However, the molecular mechanisms underlying the diversity of the level of the opioid receptor and the opioid sensitivity among individuals remain to be elucidated. In the present study, we analyzed the opioid-receptor genes of CXBK recombinant-inbred mice, which show reduced sensitivity to opioids. Northern blotting, nucleotide sequencing, and in situ hybridization histochemical analyses demonstrated that CXBK mice possessed mu-OR mRNA with a normal coding region but an abnormally long untranslated region (UTR). In addition, the mu-OR mRNA level in CXBK mice was less than in the control mice. Next, we produced littermate mice that had inherited two copies of the wild-type mu-OR gene, had inherited two copies of the CXBK mu-OR gene, and had inherited both copies of the mu-OR genes. In these mice, inheritance of the CXBK mu-OR gene was well correlated with less mu-OR mRNA and reduced opioid effects on nociception and locomotor activity. We conclude that the CXBK mu-OR gene is responsible for the CXBK phenotypes. Because UTR differences are known to affect the level of the corresponding mRNA and protein and because UTRs are more divergent among individuals than coding regions, the present findings suggest that opioid sensitivity may vary, depending on different mu-OR levels attributable to divergent UTR of mu-OR mRNA.

A murine model of opioid-induced hyperalgesia

Controversies surround the possible long-term physiological and psychological consequences of opioid use. Analgesic tolerance and addiction are commonly at the center of these controversies, but other concerns exist as well. A growing body of evidence suggests that hyperalgesia caused by the chronic administration of opioids can occur in laboratory animals and in humans. In these studies we describe a murine model of opioid-induced hyperalgesia (OIH). After the treatment of mice for 6 days with implanted morphine pellets followed by their removal, both thermal hyperalgesia and mechanical allodynia were documented. Additional experiments demonstrated that prior morphine treatment also increased formalin-induced licking behavior. These effects were intensified by intermittent abstinence accomplished through administration of naloxone during morphine treatment. Experiments designed to determine if the μ-opioid receptor mediated OLH in our model revealed that the relatively-selective μ-opioid receptor agonist fentanyl induced the thermal hyperalgesia and mechanical allodynia characteristic of OIH when administered in intermittent boluses over 6 days. In complimentary experiments we found that CXBK mice which have reduced μ-opioid receptor binding displayed no significant OIH after morphine treatment. Finally, we explored the pharmacological sensitivities of OIH. We found that the N-methyl-d-aspartate (NMDA) receptor antagonist MK-801, the nitric oxide synthase (NOS) inhibitor NG-nitro-l-arginine methyl ester (l-NAME) and the heme oxygenase (HO) inhibitor tin protoporphyrin (Sn-P) dose-dependently reduced OIH in this model while the NSAID indomethacin had no effect. Thus we have characterized a murine model of OIH which will be useful in the pursuit of the molecular mechanisms underlying this phenomenon.

Differential involvement of μ 1-opioid receptors in endomorphin- and β-endorphin-induced G-protein activation in the mouse pons/medulla

Several genetic mouse models of differential sensitivity to opioids have been used to investigate the mechanisms underlying individual variation in responses to opioids. The CXBK mice are inbred recombinant mice which have a lower level of μ 1-opioid receptors than their parental strain. Endomorphin-1 and endomorphin-2 are endogenous opioid peptides that are highly selective for μ-opioid receptors, while β-endorphin, which is also an endogenous opioid peptide, is non-selective for μ-, δ- and putative ε-opioid receptors. The present study was designed to investigate the effects of these endogenous opioid peptides on G-protein activation by monitoring guanosine-5′- o-(3-[ 35S]thio)triphosphate binding to pons/medulla membranes of CXBK mice and their parental strain C57BL/6ByJ mice. Endomorphin-1 (0.1–10 μM), endomorphin-2 (0.1–10 μM) and β-endorphin (0.1–10 μM) increased guanosine-5′- o-(3-[ 35S]thio)triphosphate binding to the pons/medulla membranes from C57BL/6ByJ and CXBK mice in a concentration-dependent manner. However, the increases of guanosine-5′- o-(3-[ 35S]thio)triphosphate binding induced by either endomorphin-1 or endomorphin-2 in CXBK mice were significantly much lower than those in C57BL/6ByJ mice. However, no significant difference was found in the increases of the guanosine-5′- o-(3-[ 35S]thio)triphosphate binding induced by β-endorphin in C57BL/6ByJ and CXBK mice. Moreover, whereas the increase of guanosine-5′- o-(3-[ 35S]thio)triphosphate binding induced by 10 μM endomorphin-1 or endomorphin-2 were almost completely blocked by a μ-opioid receptor antagonist β-funaltrexamine (10 μM) in both strains, the increase of guanosine-5′- o-(3-[ 35S]thio)triphosphate binding induced by 10 μM β-endorphin was attenuated to approximately 70% of stimulation by co-incubation with 10 μM β-funaltrexamine in both strains. The residual stimulation of [ 35S]guanosine-5′- o-(3-thio)triphosphate binding by 10 μM β-endorphin in the presence of 10 μM β-funaltrexamine was further attenuated by the addition of putative ε-opioid receptor partial agonist β-endorphin (1–27) (1 μM) in both strains. Like the endomorphins, the synthetic μ-opioid receptor agonist [ d-Ala 2, N-MePhe 4,Gly-ol 5]enkephalin at 10 μM showed lower increases of guanosine-5′- o-(3-[ 35S]thio)triphosphate binding in CXBK mice than those in C57BL/6ByJ mice. However, there was no strain difference in the stimulation of guanosine-5′- o-(3-[ 35S]thio)triphosphate binding induced by 10 μM of the selective δ 1-opioid receptor agonist [ d-Pen 2,5]enkephalin, δ 2-opioid receptor agonist [ d-Ala 2]deltorphin II or κ-opioid receptor agonist U50,488H. The results indicate that the G-protein activation by endomorphin-1 and endomorphin-2 in the mouse pons/medulla is mediated by both μ 1- and μ 2-opioid receptors. Moreover, β-endorphin-induced G-protein activation in the mouse pons/medulla is, in part, mediated by μ 2- and putative ε-, but not by μ 1-opioid receptors.

Opioid receptor upregulation in μ-opioid receptor deficient CXBK and outbred Swiss Webster mice

Chronic in vivo treatment with opioid antagonists increases opioid receptor density and the potency of opioid agonists without altering receptor mRNA levels. To determine if basal receptor density affects opioid receptor upregulation, we examined the effect of chronic naltrexone treatment on μ-opioid receptor density and mRNA in two mice strains that differ in μ-opioid receptor density. CXBK mice (μ-opioid receptor deficient) and outbred Swiss Webster mice were implanted s.c. with a placebo or 15 mg naltrexone pellet for 8 days, the pellets removed and 24 hr later opioid receptor density (μ, δ) and receptor mRNA level (u) determined in whole brain; or morphine dose-response studies conducted. In placebo-treated CXBK mice, μ-opioid receptor density was ≈40% less than in Swiss Webster mice, although μ-opioid receptor mRNA abundance was similar in both strains. In placebo-treated CXBK mice, morphine potency was ≈6-fold less than Swiss Webster mice. Naltrexone treatment increased morphine potency (1.7-fold) and μ-(≈90%) and δ- (≈20–40%) opioid receptor density in CXBK and Swiss Webster mouse brain similarly. μ-opioid receptor mRNA was unchanged by naltrexone treatment in either strain. There was no difference in the basal or naltrexone-treated whole brain G iα2 protein levels in CXBK or Swiss Webster mouse. These data indicate that a deficiency in μ-opioid receptors does not alter the regulation of opioid receptors by opioid antagonists in vivo, and suggest that adaptive responses to chronic opioid antagonist treatment are independent of opioid receptor density.

Differences in delta opioid receptor antinociception, binding, and mRNA levels between BALB/c and CXBK mice

Mu and delta opioid receptors have been demonstrated to mediate supraspinal opioid antinociception. Whereas the recombinant inbred CXBK mouse is notably deficient in mu opioid receptor antinociception, binding density, and mRNA (MOR-1) levels, little is known about delta opioid receptor processes in this strain. The present study thus compared CXBK mice and their BALB/c strain progenitors with respect to delta opioid antinociception, whole-brain receptor binding levels, and mRNA (DOR-1) levels. Following intracerebroventricular injections of the selective delta 1 and delta 2 opioids DPDPE and [ d-Ala 2]deltorphin II, respectively, CXBK mice displayed relatively lower antinociception on the tail-flick test, resulting in significantly increased ED 50 values for both agonists in this strain. Decreased whole-brain specific binding of [ 3 H ][ d-Ala 2]deltorphin II, but not [ 3 H ]DPDPE, was also observed in CXBK mice. Solution hybridization with a probe for the DOR-1 revealed increased transcript levels in the caudate-putamen, frontal cortex, and spinal cord of this strain. The present data demonstrate a deficiency in delta 1 and delta 2 opioid antinociception in CXBK mice concomitant with reductions in whole-brain delta 2 receptor binding and regional increases in DOR-1. Whether these observations are causally related remains to be clarified.

Methadone analgesia in morphine-insensitive CXBK mice

Methadone, a potent opioid analgesic, has long been considered a μ-opioid, based upon the similarities between its actions and those of morphine. This classification is supported by the sensitivity of methadone analgesia to the highly μ-opioid receptor-selective antagonist β-funaltrexamine. Yet, CXBK mice respond normally to methadone despite their insensitivity to systemic morphine, distinguishing between the receptor mechanisms of the two drugs. β-Funaltrexamine antagonizes methadone analgesia in CXBK mice, implying that the opioid is still acting through a μ-opioid receptor. These results reveal distinct analgesic mechanisms for morphine and methadone and provide further support for multiple subtypes of μ-opioid receptors.

Immunodominant CD4+ T cell receptor Vbeta repertoires involved in graft-versus-host disease responses to minor histocompatibility antigens.

In vitro CTL responses to multiple minor histocompatibility Ags (miHA) are governed by immunodominance as demonstrated in the C57BL/6By (B6) anti-BALB.B strain combination. Immunodominance was also demonstrated to be operative in graft-vs-host disease (GVHD) responses against BALB.B-derived miHA following transplantation of B6 T cells into irradiated recipients of both the BALB.B and CXB recombinant inbred strains. The hierarchy of in vivo and in vitro T cell responses to miHA differed. GVHD did not develop in CXBG and CXBK mice, which express immunodominant miHA for CTL generation, whereas disease occurred in the BALB.B, CXBE, CXBI, and CXBJ mouse strains. Previous results demonstrated that B6 CD4+ T cells provide helper function for CD8+ T cells involved in GVHD responses in the BALB.B, CXBE, and CXBI strains. CD4+ T cells alone were mediators of GVHD in all strains except CXBE. This study analyzed the TCR Vbeta repertoires of CD4+ thoracic duct lymphocytes (TDL) collected during the initial stages of GVHD in the B6-->BALB.B and B6-->CXBE strain combinations. Positively selected CD4+ TDL from the B6-->BALB.B (B6(+BALB.B)) combination exhibited marked expansion in the TCR Vbeta6+ and Vbeta8.1/8.2+ families, as well as smaller increases in the Vbeta7 and Vbeta9 families. CD4+ TDL from the B6-->CXBE (B6(+CXBE)) combination displayed expansions in only the Vbeta7+ and Vbeta9+ families. These data suggest that B6 CD4+ T cells can recognize a limited number of immunodominant miHA during GVHD induction and that in both BALB.B and CXBE recipients, the TCR Vbeta repertoires partially overlap.