Biomedical subjects

B L Kieffer

Publications and source records attributed to B L Kieffer.

At least 37 records · Page 2Linked to original sources

PubMed2001

Immunosuppression by delta-opioid antagonist naltrindole: delta- and triple mu/delta/kappa-opioid receptor knockout mice reveal a nonopioid activity.

The delta-opioid antagonist naltrindole has been shown to inhibit graft rejection in vivo and suppress allogeneic mixed lymphocyte reaction (MLR) in vitro, similarly to cyclosporin A. We investigated whether this action is mediated by delta-opioid receptors using both genetic and pharmacological tools. Naltrindole and two related compounds, 7-benzylidene-7-dehydronaltrexone and naltriben, inhibited MLR performed with lymphocytes from wild-type and delta-opioid receptor knockout mice, with comparable potency. Furthermore, these compounds suppressed the proliferation of spleen cells from triple delta/mu/kappa-opioid receptor-deficient animals as well. Finally, the highly delta-selective, but structurally distinct, antagonist N,N-dimethyl-Dmt-Tic-OH and the general opioid antagonist naltrexone were inactive in the MLR assay. In conclusion, we demonstrate for the first time that the immunosuppressive activity of naltrindole and close derivatives is not mediated by any of the three cloned opioid receptors. Therefore, the postulated inhibitory activity of naltrindole in the graft rejection process is mediated by a target, which remains to be discovered.

Animals↗

PubMed2001

Increased ethanol self-administration in delta-opioid receptor knockout mice.

BACKGROUND: The role of the delta-opioid receptor in ethanol drinking has remained unclear despite the use of traditional pharmacological and correlational approaches. The results of several studies suggest that pharmacological blockade of these receptors results in decreases in ethanol drinking behavior, but an approximately equal number of reports have failed to observe an effect of delta-receptor antagonism on ethanol drinking. It is clear that alternative approaches to understanding opioid-receptor involvement in ethanol drinking are needed. METHODS: In this study, ethanol drinking was examined in delta-opioid receptor knockout (KO) mice by using first a two-bottle-choice test, then an operant self-administration paradigm and a second two-bottle-choice test, in that order. In addition, because KO mice were previously shown to display enhanced anxiety-like behavior relative to wild-type (WT) mice, the effect of ethanol self-administration on anxiety-like responses was determined. RESULTS: delta KO mice initially showed no evidence of a preference for ethanol in the first two-bottle-choice drinking test; however, after an experience of operant self-administration of ethanol, a preference for ethanol developed in the second two-bottle-choice test. KO mice also showed a preference for ethanol over water and self-administered more ethanol than WT mice in the operant self-administration paradigm. The ethanol self-administered in this procedure was sufficient to reverse the innate anxiety-like response observed in this strain. CONCLUSIONS: delta KO mice showed a greater preference for ethanol and consumed more ethanol than their WT counterparts, suggesting that a decrease in delta-receptor activity is associated with increased ethanol-drinking behavior. It is hypothesized that delta receptors may influence ethanol self-administration at least partly through an effect of these receptors on anxiety-like behavior.

Animals↗

PubMed2000

In vitro pharmacological profile of peptide III-BTD: a novel ligand for nociceptin/orphanin FQ and opioid receptors.

Peptide III-BTD has been recently identified as a non-selective nociceptin/orphanin FQ receptor ligand by screening of a synthetic peptide combinatorial library. In the present study we evaluated the pharmacological profile of peptide III-BTD in isolated tissues (mouse and rat vas deferens, guinea pig ileum) sensitive to both nociceptin and opioid peptides. In the mouse vas deferens and guinea pig ileum, III-BTD concentration dependently inhibited the electrically induced twitch (pEC50 5.91 and 6.18, respectively; Emax 94 +/- 1% and 94 +/- 2%) and this effect was blocked by naloxone (1 microM). In the rat vas deferens, III-BTD was inactive in most of the tissues, while in few others it elicited a slight inhibition only at the highest concentration tested (10 microM). In the presence of 1 microM naloxone, 1 microM III-BTD shifted to the right the concentration response curve of nociceptin in a parallel manner, showing pKB values in the range 6.6-6.9. These data confirm on native nociceptin receptors the pharmacological profile of peptide III-BTD which behaved as a mixed nociceptin receptor antagonist/opioid receptor agonist in the [35S]GTPyS binding assay performed on cells expressing the recombinant human receptors.

Animals↗

PubMed2000

A single nucleotide polymorphic mutation in the human mu-opioid receptor severely impairs receptor signaling.

Large scale sequencing of the human mu-opioid receptor (hMOR) gene has revealed polymorphic mutations that occur within the coding region. We have investigated whether the mutations N40D in the extracellular N-terminal region, N152D in the third transmembrane domain, and R265H and S268P in the third intracellular loop alter functional properties of the receptor expressed in mammalian cells. The N152D receptor was produced at low densities. Binding affinities of structurally diverse opioids (morphine, diprenorphine, DAMGO and CTOP) and the main endogenous opioid peptides (beta-endorphin, [Met]enkephalin, and dynorphin A) were not markedly changed in mutant receptors (<3-fold). Receptor signaling was strongly impaired in the S268P mutant, with a reduction of efficacy and potency of several agonists (DAMGO, beta-endorphin, and morphine) in two distinct functional assays. Signaling at N40D and R265H mutants was highly similar to wild type, and none of the mutations induced detectable constitutive activity. DAMGO-induced down-regulation of receptor-binding sites, following 20 h of treatment, was identical in wild-type and mutant receptors. Our data show that natural sequence variations in hMOR gene have little influence on ligand binding or receptor down-regulation but could otherwise modify receptor density and signaling. Importantly, the S268P mutation represents a loss-of-function mutation for the human mu-opioid receptor, which may have an incidence on opioid-regulated behaviors or drug addiction in vivo.

Analgesics, Opioid↗

PubMed2000

Mice deficient for delta- and mu-opioid receptors exhibit opposing alterations of emotional responses.

The role of the opioid system in controlling pain, reward and addiction is well established, but its role in regulating other emotional responses is poorly documented in pharmacology. The mu-, delta- and kappa- opioid receptors (encoded by Oprm, Oprd1 and Oprk1, respectively) mediate the biological activity of opioids. We have generated Oprd1-deficient mice and compared the behavioural responses of mice lacking Oprd1, Oprm (ref. 6) and Oprk1 (ref. 7) in several models of anxiety and depression. Our data show no detectable phenotype in Oprk1-/- mutants, suggesting that kappa-receptors do not have a role in this aspect of opioid function; opposing phenotypes in Oprm-/- and Oprd1-/- mutants which contrasts with the classical notion of similar activities of mu- and delta-receptors; and consistent anxiogenic- and depressive-like responses in Oprd1-/- mice, indicating that delta-receptor activity contributes to improvement of mood states. We conclude that the Oprd1-encoded receptor, which has been proposed to be a promising target for the clinical management of pain, should also be considered in the treatment of drug addiction and other mood-related disorders.

Animals↗

PubMed2000

Specific activation of the mu opioid receptor (MOR) by endomorphin 1 and endomorphin 2.

The recently discovered endomorphin 1 (Tyr-Pro-Trp-Phe-NH2) and endomorphin 2 (Tyr-Pro-Phe-Phe-NH2) were investigated with respect to their direct receptor-binding properties, and to their ability to activate G proteins and to inhibit adenylyl cyclase in both cellular and animal models. Both tetrapeptides activated G proteins and inhibited adenylyl cyclase activity in membrane preparations from cells stably expressing the mu opioid receptor, an effect reversed by the mu receptor antagonist CTAP (D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2), but they had no influence on cells stably expressing the delta opioid receptor. To further establish the selectivity of these peptides for the mu opioid receptor, brain preparations of mice lacking the mu opioid receptor gene were used to study their binding and signalling properties. Endomorphin 2, tritiated by a dehalotritiation method resulting in a specific radioactivity of 1.98 TBq/mmol (53.4 Ci/mmol), labelled the brain membranes of wild-type mice with a Kd value of 1.77 nM and a Bmax of 63.33 fmol/mg protein. In membranes of mice lacking the mu receptor gene, no binding was observed, and both endomorphins failed to stimulate [35S]guanosine-5'-O-(3-thio)triphosphate ([35S]GTPgammaS) binding and to inhibit adenylyl cyclase. These data show that endomorphins are capable of activating G proteins and inhibiting adenylyl cyclase activity, and all these effects are mediated by the mu opioid receptors.

Adenylyl Cyclases↗

PubMed2000

Opioid receptors: from genes to mice.

Opiates produce their strong analgesic and addictive actions by activating mu-, delta-, and kappa-opioid receptors, which otherwise interact with endogenous opioid peptides to regulate nociception, mood, and responses to stress. The recent cloning of an opioid receptor gene family has allowed the production of null-mutant mice for each mu-, delta-, and kappa-receptor gene. Initial observation of receptor-deficient mice shows no obvious developmental abnormality, and reveals subtle modifications of pain perception in adult mice. Pharmacologic responses to standard opiates have been evaluated carefully, and results suggest that morphine produces its main biologic actions by acting specifically at mu-receptors, whereas delta-agonists seem weakly delta-selective under in vivo experimental conditions. Future studies of single- and combinatorial-opioid receptor-deficient mice will clarify the specific role of each receptor in chronic pain, motivation, and addictive behaviors. These mice also represent useful tools in the development of novel opioid compounds of therapeutic interest.

Journal Article↗

PubMed2000

mu-Opioid receptor knockout mice do not self-administer alcohol.

Opioid peptides long have been hypothesized to play a role in ethanol reinforcement. Neuropharmacological studies have shown that opioid receptor antagonists decrease ethanol self-administration in rodents and prevent relapse in humans. However, the exact mechanism for such powerful effects has remained elusive. The availability of mu-opioid receptor knockout mice has made possible the direct examination of the role of the mu-opioid receptor in mediating ethanol self-administration. In the present experiments, both nosepoke and lever operant ethanol self-administration and several tests of two bottle-choice ethanol drinking were studied in these genetically engineered mice. In no case did knockout mice show evidence of ethanol self-administration, and, in fact, these mice showed evidence of an aversion to ethanol under several experimental conditions. These data provide new evidence for a critical role for mu-opioid receptors in ethanol self-administration assessed with a variety of behavioral paradigms and new insights into the neuropharmacological basis for ethanol reinforcement.

Alcohol Drinking↗

PubMed1999

Ligands for kappa-opioid and ORL1 receptors identified from a conformationally constrained peptide combinatorial library.

We have screened a synthetic peptide combinatorial library composed of 2 x 10(7) beta-turn-constrained peptides in binding assays on four structurally related receptors, the human opioid receptors mu, delta, and kappa and the opioid receptor-like ORL1. Sixty-six individual peptides were synthesized from the primary screening and tested in the four receptor binding assays. Three peptides composed essentially of unnatural amino acids were found to show high affinity for human kappa-opioid receptor. Investigation of their activity in agonist-promoted stimulation of [(35)S]guanosine 5'-3-O-(thio)triphosphate binding assay revealed that we have identified the first inverse agonist as well as peptidic antagonists for kappa-receptors. To fine-tune the potency and selectivity of these kappa-peptides we replaced their turn-forming template by other turn mimetic molecules. This "turn-scan" process allowed the discovery of compounds with modified selectivity and activity profiles. One peptide displayed comparable affinity and partial agonist activity toward all four receptors. Interestingly, another peptide showed selectivity for the ORL1 receptor and displayed antagonist activity at ORL1 and agonist activity at opioid receptors. In conclusion, we have identified peptides that represent an entirely new class of ligands for opioid and ORL1 receptors and exhibit novel pharmacological activity. This study demonstrates that conformationally constrained peptide combinatorial libraries are a rich source of ligands that are more suitable for the design of nonpeptidal drugs.

Amino Acid Sequence↗

PubMed1999

Constitutive activation of the delta opioid receptor by mutations in transmembrane domains III and VII.

We have investigated whether transmembrane amino acid residues Asp128 (domain III), Tyr129 (domain III) [corrected], and Tyr308 (domain VII) in the mouse delta opioid receptor play a role in receptor activation. To do so, we have used a [35S]GTPgammaS (where GTPgammaS is guanosine 5'-3-O-(thio)triphosphate) binding assay to quantify the activation of recombinant receptors transiently expressed in COS cells and compared functional responses of D128N, D128A, Y129F, Y129A, and Y308F point-mutated receptors to that of the wild-type receptor. In the absence of ligand, [35S]GTPgammaS binding was increased for every mutant receptor under study (1.6-2.6-fold), suggesting that all mutations are able to enhance constitutive activity at the receptor. In support of this finding, the inverse agonist N,N-diallyl-Tyr-Aib-Aib-Phe-Leu (where Aib represents alpha-aminobutyric acid) efficiently reduced basal [35S]GTPgammaS binding in the mutated receptor preparations. The potent agonist BW373U86 stimulated [35S]GTPgammaS binding above basal levels with similar (D128N, Y129F, and Y129A) or markedly increased (Y308F) efficacy compared with wild-type receptor. BW373U86 potency was maintained or increased. In conclusion, our results demonstrate that the mutations under study increase functional activity of the receptor. Three-dimensional modeling suggests that Asp128 (III) and Tyr308 (VII) interact with each other and that Tyr129 (III) undergoes H bonding with His278 (VI). Thus, Asp128, Tyr129, and Tyr308 may be involved in a network of interhelical bonds, which contributes to maintain the delta receptor under an inactive conformation. We suggest that the mutations weaken helix-helix interactions and generate a receptor state that favors the active conformation and/or interacts with heterotrimeric G proteins more effectively.

Amino Acid Substitution↗

PubMed1999

Opioids: first lessons from knockout mice.

Opioid receptors of the mu-, delta- and kappa-subtypes mediate the potent analgesic and addictive actions of opioid drugs. They also regulate responses to pain, stress and emotions when activated by endogenous opioid peptides. Recently, mice lacking opioid receptors or opioid peptides have been produced by gene targeting, providing molecular tools to study opioid function in vivo. Observations on mutant mice have shed new light on the mode of action of opioids, opioid receptor heterogeneity and interactions, and the involvement of each component of the opioid system in mouse physiology. In this article, Brigitte L. Kieffer reviews the first reported studies and discusses their therapeutic implications.

Animals↗

PubMed1999

Expression of delta, kappa and mu human opioid receptors in Escherichia coli and reconstitution of the high-affinity state for agonist with heterotrimeric G proteins.

Human opioid receptors of the delta, mu and kappa subtypes were successfully expressed in Escherichia coli as fusions to the C-terminus of the periplasmic maltose-binding protein, MBP. Expression levels of correctly folded receptor molecules were comparable for the three subtypes and reached an average of 30 receptors.cell-1 or 0.5 pmol.mg-1 membrane protein. Binding of [3H]diprenorphine to intact cells or membrane preparations was saturatable, with a dissociation constant, KD, of 2.5 nM, 0.66 nM and 0.75 nM for human delta, mu and kappa opioid receptors (hDOR, hMOR and hKOR, respectively). Recombinant receptors of the three subtypes retained selectivity and nanomolar affinity for their specific antagonists. Agonist affinities were decreased by one to three orders of magnitude as compared to values measured for receptors expressed in mammalian cells. The effect of sodium on agonist binding to E. coli-expressed receptors was investigated. Receptor high-affinity state for agonists was reconstituted in the presence of heterotrimeric G proteins. We also report affinity values of endomorphins 1 and 2 for mu opioid receptors expressed both in E. coli and in COS cells. Our results confirm that opioid receptors can be expressed in a functional form in bacteria and point out the advantages of E. coli as an expression system for pharmacological studies.

Animals↗

PubMed1998

Activity of the delta-opioid receptor is partially reduced, whereas activity of the kappa-receptor is maintained in mice lacking the mu-receptor.

Previous pharmacological studies have indicated the possible existence of functional interactions between mu-, delta- and kappa-opioid receptors in the CNS. We have investigated this issue using a genetic approach. Here we describe in vitro and in vivo functional activity of delta- and kappa-opioid receptors in mice lacking the mu-opioid receptor (MOR). Measurements of agonist-induced [35S]GTPgammaS binding and adenylyl cyclase inhibition showed that functional coupling of delta- and kappa-receptors to G-proteins is preserved in the brain of mutant mice. In the mouse vas deferens bioassay, deltorphin II and cyclic[D-penicillamine2, D-penicillamine5] enkephalin exhibited similar potency to inhibit smooth muscle contraction in both wild-type and MOR -/- mice. delta-Analgesia induced by deltorphin II was slightly diminished in mutant mice, when the tail flick test was used. Deltorphin II strongly reduced the respiratory frequency in wild-type mice but not in MOR -/- mice. Analgesic and respiratory responses produced by the selective kappa-agonist U-50,488H were unchanged in MOR-deficient mice. In conclusion, the preservation of delta- and kappa-receptor signaling properties in mice lacking mu-receptors provides no evidence for opioid receptor cross-talk at the cellular level. Intact antinociceptive and respiratory responses to the kappa-agonist further suggest that the kappa-receptor mainly acts independently from the mu-receptor in vivo. Reduced delta-analgesia and the absence of delta-respiratory depression in MOR-deficient mice together indicate that functional interactions may take place between mu-receptors and central delta-receptors in specific neuronal pathways.

3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cycloh↗

PubMed1998

Abolition of morphine-immunosuppression in mice lacking the mu-opioid receptor gene.

Opiates are potent analgesic and addictive compounds. They also act on immune responses, and morphine, the prototypic opiate, has been repeatedly described as an immunosuppressive drug. Pharmacological studies have suggested that the inhibitory action of opiates on immunity is mediated by multiple opioid receptor sites but molecular evidence has remained elusive. Recently, three genes encoding mu- (MOR), delta-, and kappa-opioid receptors have been cloned. To investigate whether the mu-opioid receptor is functionally implicated in morphine immunosuppression in vivo, we have examined immune responses of mice with a genetic disruption of the MOR gene. In the absence of drug, there was no difference between wild-type and mutant mice with regard to a large number of immunological endpoints, suggesting that the lack of MOR-encoded protein has little consequence on immune status. Chronic morphine administration induced lymphoid organ atrophy, diminished the ratio of CD4(+)CD8(+) cells in the thymus and strongly reduced natural killer activity in wild-type mice. None of these effects was observed in MOR-deficient mice after morphine treatment. This demonstrates that the MOR gene product represents a major molecular target for morphine action on the immune system. Because our previous studies of MOR-deficient mice have shown that this receptor protein is also responsible for morphine analgesia, reward, and physical dependence, the present results imply that MOR-targeted therapeutic drugs that are developed for the treatment of pain or opiate addiction may concomitantly influence immune responses.

Animals↗

PubMed1998

Disruption of the kappa-opioid receptor gene in mice enhances sensitivity to chemical visceral pain, impairs pharmacological actions of the selective kappa-agonist U-50,488H and attenuates morphine withdrawal.

***micro***-, delta- and kappa-opioid receptors are widely expressed in the central nervous system where they mediate the strong analgesic and mood-altering actions of opioids, and modulate numerous endogenous functions. To investigate the contribution of the kappa-opioid receptor (KOR) to opioid function in vivo, we have generated KOR-deficient mice by gene targeting. We show that absence of KOR does not modify expression of the other components of the opioid system, and behavioural tests indicate that spontaneous activity is not altered in mutant mice. The analysis of responses to various nociceptive stimuli suggests that the KOR gene product is implicated in the perception of visceral chemical pain. We further demonstrate that KOR is critical to mediate the hypolocomotor, analgesic and aversive actions of the prototypic kappa-agonist U-50, 488H. Finally, our results indicate that this receptor does not contribute to morphine analgesia and reward, but participates in the expression of morphine abstinence. Together, our data demonstrate that the KOR-encoded receptor plays a modulatory role in specific aspects of opioid function.

3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cycloh↗

PubMed1998

Distribution of nociceptin/orphanin FQ receptor transcript in human central nervous system and immune cells.

We have examined the distribution of the opioid receptor-like-1 (ORL-1) transcript in the human CNS as well as human immune cells by RT-PCR and RNAse protection. The hORL-1 mRNA was distributed throughout the brain and particularly abundant in cortical areas, striatum, thalamus and hypothalamus. In the immune system, gene transcription was observed in normal circulating lymphocytes and monocytes as well as in T, B and monocytic cell lines. A splice variant, lacking 15 nucleotides at the junction between exon 1 and exon 2, showed a distribution similar to the already known ORL-1 transcript. Altogether these results show comparable expression levels of the hORL-1 gene in both nervous and immune systems, suggesting that the ORL-1-encoded receptor may participate to neuronal and non-neuronal physiological functions in humans.

Adult↗

PubMed1998

A molecular basis for opiate action.

Opioid receptors mediate the strong analgesic and addictive actions of exogenous opiates, the prototype of which is morphine. The opioid system consists of a family of endogenous opioid peptides and three receptor types, m, d and k. It is widely distributed throughout the CNS and regulates a large diversity of physiological functions, including pain perception and mood control. The recent cloning of opioid receptors has opened up a new era in opioid research. The molecular basis of opioid action may now be addressed by in vitro structure-function studies of recombinant receptors and by in vivo gene targeting. Novel drug design strategies based on data obtained from molecular approaches will be developed in order to generate the long-sought non-addictive analgesic.

Alkaloids↗

PubMed1997

Mu and delta opioid receptors are differentially desensitized by the coexpression of beta-adrenergic receptor kinase 2 and beta-arrestin 2 in xenopus oocytes.

The Xenopus oocyte expression system was used to test the hypothesis that homologous opioid receptor desensitization results from receptor phosphorylation by G protein-coupled receptor kinases. Activation of delta (DOR), mu (MOR) opioid, or beta2-adrenergic receptors increased K+ conductance in oocytes coexpressing the G protein-gated inwardly rectifying K+ channel subunits GIRK1 and GIRK4, and the intrinsic rate of desensitization was small. Coexpression of beta-adrenergic receptor kinase 2 (beta-ARK2) and beta-arrestin 2 (beta-arr2) synergistically produced a rapid desensitization of both DOR and beta2-adrenergic receptor signaling with a t1/2 < 4 min. beta-ARK2 and beta-arr2 more slowly desensitized MOR responses; a similar synergistic effect on MOR required 2-3 h of agonist treatment. DOR mutants lacking serine and threonine residues at the end of the cytoplasmic tail coupled effectively to GIRK channels but were insensitive to beta-ARK2 and beta-arr2. However, a DOR mutant having serine residues mutated to alanine in the third cytoplasmic loop was indistinguishable in coupling and desensitization from the wild type DOR. These studies establish that opioid receptors can be regulated by beta-ARK2 and beta-arr2 and that a portion of the COOH terminus of DOR enhances sensitivity to this modulation.

Amino Acid Sequence↗