Bernard P. Roques

Absence of opiate rewarding effects in mice lacking dopamine D2 receptors

Dopamine receptors have been implicated in the behavioural response to drugs of abuse. These responses are mediated particularly by the mesolimbic dopaminergic pathway arising in the ventral tegmental area and projecting to the limbic system. The rewarding properties of opiates and the somatic expression of morphine abstinence have been related to changes in mesolimbic dopaminergic activity that could constitute the neural substrate for opioid addiction. These adaptive responses to repeated morphine administration have been investigated in mice with a genetic disruption of the dopaminergic D2 receptors. Although the behavioural expression of morphine withdrawal was unchanged in these mice, a total suppression of morphine rewarding properties was observed in a place-preference test. This effect is specific to the drug, as mice lacking D2 receptors behaved the same as wild-type mice when food is used as reward. We conclude that the D2 receptor plays a crucial role in the motivational component of drug addiction.

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

μ‐, δ‐ and κ‐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 κ‐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 κ‐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.

Reduction of Morphine Abstinence in Mice with a Mutation in the Gene Encoding CREB

Chronic morphine administration induces an up-regulation of several components of the cyclic adenosine 5′-monophosphate (cAMP) signal transduction cascade. The behavioral and biochemical consequences of opiate withdrawal were investigated in mice with a genetic disruption of the α and Δ isoforms of the cAMP-responsive element-binding protein (CREB). In CREBαΔ mutant mice the main symptoms of morphine withdrawal were strongly attenuated. No change in opioid binding sites or in morphine-induced analgesia was observed in these mutant mice, and the increase of adenylyl cyclase activity and immediate early gene expression after morphine withdrawal was normal. Thus, CREB-dependent gene transcription is a factor in the onset of behavioral manifestations of opiate dependence.

Structure of the winged-helix protein hRFX1 reveals a new mode of DNA binding.

Regulatory factor X (RFX) proteins are transcriptional activators that recognize X-boxes (DNA of the sequence 5′-GTNRCC(0–3N)RGYAAC-3′, where N is any nucleotide, R is a purine and Y is a pyrimidine) using a highly conserved 76-residue DNA-binding domain (DBD). DNA-binding defects in the protein RFX5 cause bare lymphocyte syndrome or major histocompatibility antigen class II deficiency. RFX1, -2 and -3 regulate expression of other medically important gene products (for example, interleukin-5 receptor α chain, IL-5Rα). Fusions of the ligand-binding domain of the oestrogen receptor with the DBD of RFX4 occur in some human breast tumours. Here we present a 1.5 Å-resolution structure of two copies of the DBD of human RFX1 (hRFX1) binding cooperatively to a symmetrical X-box. hRFX1 is an unusual member of the winged-helix subfamily of helix–turn–helix proteins because it uses a β-hairpin (or wing) to recognize DNA instead of the recognition helix typical of helix–turn–helix proteins. A new model for interactions between linker histones and DNA is proposed.

Behavioural and biochemical evidence for signs of abstinence in mice chronically treated with Δ-9-tetrahydrocannabinol

Tolerance and dependence induced by chronic Δ‐9‐tetrahydrocannabinol (THC) administration were investigated in mice. The effects on body weight, analgesia and hypothermia were measured during 6 days of treatment (10 or 20 mg kg−1 THC twice daily). A rapid tolerance to the acute effects was observed from the second THC administration. The selective CB‐1 receptor antagonist SR 141716A (10 mg kg−1) was administered at the end of the treatment, and somatic and vegetative manifestations of abstinence were evaluated. SR 141716A administration precipitated several somatic signs that included wet dog shakes, frontpaw tremor, ataxia, hunched posture, tremor, ptosis, piloerection, decreased locomotor activity and mastication, which can be interpreted as being part of a withdrawal syndrome. Brains were removed immediately after the behavioural measures and assayed for adenylyl cyclase activity. An increase in basal, forskolin and calcium/calmodulin stimulated adenylyl cyclase activities was specifically observed in the cerebellum of these mice. The motivational effects of THC administration and withdrawal were evaluated by using the place conditioning paradigm. No conditioned change in preference to withdrawal associated environment was observed. In contrast, a conditioned place aversion was produced by the repeated pairing of THC (20 mg kg−1), without observing place preference at any of the doses used. This study constitutes a clear behavioural and biochemical model of physical THC withdrawal with no motivational aversive consequences. This model permits an easy quantification of THC abstinence in mice and can be useful for the elucidation of the molecular mechanisms involved in cannabinoid dependence.

Analgesic effects of kelatorphan, a new highly potent inhibitor of multiple enkephalin degrading enzymes

Kelatorphan, [(R)-3-(N-hydroxy)-carboxamido-2-benzylpropanoyl]-L-alanine, represents the first virtually complete inhibitor of enkephalins metabolism with KI = 1.4 nM against enkephalinase, KI = 2 nM against the Gly2 -Gly3 cleaving dipeptidylaminopeptidase and KI = 7 microM on aminopeptidase activity. The analgesic effect of [Met5]enkephalin was potentiated 50000 times (ED50 approximately 10 ng) by intracerebroventricular co-administration in mice of kelatorphan (50 micrograms). This effect was significantly higher than that produced by bestatin (50 micrograms) + thiorphan (50 micrograms). Kelatorphan alone was at least two-fold more potent as analgesic than the above mixture of inhibitors.

D-Tyr—Ser—Gly—Phe—Leu—Thr, a highly preferential ligand for δ-opiate receptors

Binding studies of radiolabeled enkephalins with brain homogenates [l-3] suggest that these peptides interact with at least two distinct classes of binding sites called p and S receptors. Enkephalins bind to 6 receptors which constitute almost 20% of the total binding with a high affinity (Kd _ 0.5 nM and to &f receptors with a lower af~nity (Kd 5 nM) [ 1,2]. Furthermore, displacement experiments with antagonists show that morphine and synthetic opiates interact strongly with g receptors whereas peptides exhibit a high preference for 6 receptors [ 1,2,4]. Such results were also found in peripheric organs and attributed to the preponderance of g receptors in the guinea pig ileum (GPI) and 6 receptors in the mouse vas deferens (MVD) [l-4]. Therefore, the difference in the pharmacological action of any compound on the two organs was used to evaluate its p and 6 specificity [4]. The biological significance of the presence of +U and 6 receptors in the brain remains still unknown although it seems that p receptors could be involved in analgesia [4] whereas 6 receptors might be implicated in behavioral effects [S] . A possible dissociation of the pharmacological responses associated to multiple opiate receptors have incited a great number of studies in vivo [6.173 and in vitro [3,7] for the last months. However. these studies were perfo~ed with D-Ala’-D-LeuS enkephalin (DADL), a compound which exhibits only a partial specificity for 6 receptors 141. Therefore it was of great importance to search for a much higher specific ligand, in order to avoid multiple biological responses arising from simultaneous binding to the two kinds of states. Starting from conformational data and structure activity relationships in the enkephalin series, such a compound was prepared. This new hexapeptide Tyr-D-SerGly-Phe-Leu-Thr exhibits a very high specificity for 6 receptor, since its potency is 620-times greater in MVD (f& = 0.58 nM) than in GPI (K’,, = 360 nM).

Deltakephalin, Tyr-D-Thr-Gly-Phe-Leu-Thr: A new highly potent and fully specific agonist for opiate δ-receptors

Deltakephalin, Tyr-D-Thr-Gly-Phe-Leu-Thr (DTLET) was rationally designed as pure delta-probe from proposed models of mu and delta opiate receptors. On peripheral organs, deltakephalin displays a 3000 times higher inhibitory potency on the electrically stimulated mouse vas deferens (IC50 = 0.15 nM) as on the guinea pig ileum (IC50 = 460 nM). As expected [3H]deltakephalin interacts at 35 degrees C in rat brain tissue to a single class of binding sites (delta) (Bmax = 0.115 pmole/mg protein) with a high affinity: KD = 1.35 nM from equilibrium measurements and KD = 0.43 nM from kinetic determinations. Deltakephalin occurs as the most specific ligand for delta-binding sites as shown by the following discrimination ratios KI(mu)/KI(delta): 0.31 for D-Ala2-D-Leu5-enkephalin; 0.15 for D-Ser2-Thr6-Leu-enkephalin and 0.05 for deltakephalin.

Conformation of Met5-enkephalin determined by high field PMR spectroscopy.

THE discovery by Hughes et al.1 that peptides (the enkephalins) could be the endogenous agonists of opiates in brain represents a challenge to medical chemistry both in its fundamental aspects and for the possible development of new therapeutic approaches. To understand how non-peptidic substances belonging to various chemical classes can mimic the action of the pentapeptide enkephalins it is first necessary to determine the spatial arrangement of the latter. We have thus synthesised large quantities of Met5-enkephalin (H–Tyr–Gly–Gly–Phe–Met–OH) and determined its preferential conformation in solution using high field proton magnetic resonance (PMR) spectroscopy at variable temperatures, coupling constants in relation with Karplus–Bystrov curves2 and conformational energy steric maps3.

Sequential involvement of Cdk1, mTOR and p53 in apoptosis induced by the HIV‐1 envelope

Syncytia arising from the fusion of cells expressing the HIV‐1‐encoded Env gene with cells expressing the CD4/CXCR4 complex undergo apoptosis following the nuclear translocation of mammalian target of rapamycin (mTOR), mTOR‐mediated phosphorylation of p53 on Ser15 (p53S15), p53‐dependent upregulation of Bax and activation of the mitochondrial death pathway. p53S15 phosphorylation is only detected in syncytia in which nuclear fusion (karyogamy) has occurred. Karyogamy is secondary to a transient upregulation of cyclin B and a mitotic prophase‐like dismantling of the nuclear envelope. Inhibition of cyclin‐dependent kinase‐1 (Cdk1) prevents karyogamy, mTOR activation, p53S15 phosphorylation and apoptosis. Neutralization of p53 fails to prevent karyogamy, yet suppresses apoptosis. Peripheral blood mononuclear cells from HIV‐1‐infected patients exhibit an increase in cyclin B and mTOR expression, correlating with p53S15 phosphorylation and viral load. Cdk1 inhibition prevents the death of syncytia elicited by HIV‐1 infection of primary CD4 lymphoblasts. Thus, HIV‐1 elicits a pro‐apoptotic signal transduction pathway relying on the sequential action of cyclin B–Cdk1, mTOR and p53.