The effect of neuropeptide FF in the amygdala kindling model.

OBJECTIVE: Neuropeptide FF (NPFF) and its receptors (NPFF1 R and NPFF2 R) are differentially distributed throughout the central nervous system. NPFF reduces cortical excitability in rats when administered intracerebroventricularly (i.c.v.), and both NPFF and NPFF1 R antagonists attenuate pilocarpine-induced limbic seizures. In this study, our aim was to determine whether NPFF exerts anticonvulsant or anti-epileptogenic effects in the rat amygdala kindling model for temporal lobe seizures. METHODS: Male Wistar rats were implanted with a recording/stimulation electrode in the right amygdala and a cannula in the left lateral ventricle. In a first group of animals, the afterdischarge threshold (ADT) was determined after a single i.c.v. infusion of saline (n = 8) or NPFF (1 nmol/h for 2 h; n = 10). Subsequently, daily infusion of saline (n = 8) or NPFF (1 nmol/h for 2 h; i.c.v.; n = 9) was performed, followed by a kindling stimulus (ADT+200 µA). Afterdischarge duration and seizure severity were evaluated after every kindling stimulus. A second group of rats (n = 7) were fully kindled, and the effect of saline or a high dose of NPFF (10 nmol/h for 2 h, i.c.v.) on ADT and the generalized seizure threshold (GST) was subsequently determined. RESULTS: In naive rats, NPFF significantly increased the ADT compared to control (435 ± 72 µA vs 131 ± 23 µA [P < 0.05]). When rats underwent daily stimulations above the ADT, NPFF did not delay or prevent kindling acquisition. Furthermore, a high dose of NPFF did not alter ADT or GST in fully kindled rats. CONCLUSIONS: I.c.v. administration of NPFF reduced excitability in the amygdala in naive, but not in fully kindled rats, and had no effect on kindling acquisition.

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.

Zinc-binding domain of poly(ADP-ribose)polymerase participates in the recognition of single strand breaks on DNA.

Poly(ADP-ribose)polymerase is a chromatin-associated enzyme of eukaryotic cell nuclei that catalyses the covalent attachment of ADP-ribose units from NAD+ to various nuclear acceptor proteins. This post-translational modification has been postulated to influence several chromatin functions, particularly those where nicking and rejoining of DNA occur. Poly(ADP-ribosyl)ation reactions are strictly dependent upon the presence of interruptions on DNA. We have recently demonstrated that the DNA-binding domain of the protein containing two putative "zinc-fingers" binds DNA in a zinc-dependent manner. The basis for the recognition of the DNA strand breaks by this enzyme, and more precisely, its 29,000 Mr N-terminal part, which contains the metal binding sites, needed to be clarified. DNA probes harbouring a single strand interruption at a defined position were constructed from synthetic oligonucleotides. DNase I protection studies show that poly(ADP-ribose)polymerase specifically binds to a DNA single-strand break by its metal-binding domain depending upon the presence of Zn(II). These results support the idea that the enzyme participates to the maintenance of DNA integrity in eukaryotes.

Differential involvement of the mu and kappa opioid receptors in spatial learning.

In order to test the role of mu and kappa opioid receptors (Mu opioid receptor (MOR) and Kappa opioid receptor (KOR)) in hippocampal-dependent spatial learning, we analyzed genetically engineered null mutant mice missing the functional MOR or KOR gene. Compared to wild-type mice, the homozygous MOR null mutants exhibited an impairment in the ultimate level of spatial learning as shown in two distinct tasks, the 8-arm radial-maze and the Morris water-maze. Control behaviors were normal. The learning impairment could be associated with the impairment we found in the maintenance of long-term potentiation in mossy fibers in CA3. In comparison, there was no impairment in spatial learning in our KOR mutants or in mossy fibers (mf) in CA3 region long-term potentiation (LTP). Our work suggests that the MOR may play a positive role in learning and memory by increasing LTP in CA3 neurons.

Overproduction and large-scale purification of the human poly(ADP-ribose) polymerase using a baculovirus expression system.

We have overproduced the full-length human poly(ADP-ribose) polymerase (PARP) in Spodoptera frugiperda (Sf9) cells using a baculovirus expression vector system. Approx. 20 mg of purified protein from 5 x 10(8) Sf9 cells were obtained by a simple three-step purification procedure including 3-aminobenzamide affinity chromatography. The recombinant protein (rePARP), which migrates as a unique 116-kDa band on SDS-polyacrylamide gels, was identified as PARP by Western blotting using either polyclonal or monoclonal antibodies raised against the purified human and calf thymus enzymes. Furthermore, rePARP is a functional protein, as demonstrated by its ability to specifically bind Zn2+ and DNA, and to recognize single-strand breaks in DNA. The purified enzyme has the same affinity for NAD+ and turnover number as the human placental PARP. Thus, rePARP produced in insect cells is biologically active and suitable for functional analysis. The reproducibility of the overproduction and the simplicity of the purification protocol, as well as the yield of the produced protein, should greatly facilitate physicochemical and structural studies.

Identification of kappa- and delta-opioid receptor transcripts in immune cells.

To investigate the role of opioids as direct modulators of the immune response, we have searched for expression of the recently cloned delta, mu and kappa opioid receptors in immune cells. We have devised a reverse transcriptase-polymerase chain reaction strategy which specifically detects a region spanning putative transmembrane regions 2 to 7 for each transcript in both human and mouse immune cells. In human peripheral blood lymphocyte and monocyte preparations, delta was undetectable while the kappa transcript was present. The analysis of human cell lines revealed low but significant levels of delta opioid receptor transcripts in T, B or monocyte cell lines while the kappa transcript was found in B cell lines only. Investigation of murine cells showed the presence of transcript for the delta receptor in splenocytes and in some T and B cell lines. Unexpectedly, no expression of the mu receptor was detected. Sequence analysis of PCR products demonstrated nucleotide identity between immune and neuronal transcripts, indicating that they derive from the same genes. In conclusion, our results lead to the identification of kappa and delta opioid receptor transcripts in immune cells.

Expression in E. coli of the catalytic domain of rat poly(ADP-ribose)polymerase.

A 2 kilobase pair cDNA coding for the entire C-terminal catalytic domain of rat poly(ADP-ribose)polymerase has been expressed in E. coli. The overproduced 55 kDa polypeptide is active in synthesizing poly(ADP-ribose) and the 4 kDa N-terminal region of this domain is recognized by the monoclonal antibody C I,2 directed against the calf enzyme. Also, the minor alpha-chymotrypsin cleavage site found in the human catalytic domain is not present in the rat enzyme as revealed by the absence of the 40 kDa specific degradation product in the E. coli cells expressing the rat domain. The expression of this partial rat cDNA should thus permit the rapid purification and subsequent crystallization of the catalytic domain of the enzyme.

Antibody response and allogeneic mixed lymphocyte reaction in mu-, delta-, and kappa-opioid receptor knockout mice.

The implication of opioid receptors in immune response has been studied using mu-, delta- and kappa-opioid receptor knockout mice. The mutant animals were compared to their wild-type (WT) counterparts for antibody (Ab) response to the prototype Ag keyhole limpet hemocyanin (KLH). Kappa-receptor deficient mice displayed higher Ab titers for either total Ig, IgM, IgG1 or IgG2a isotypes, whereas mu and delta animals behaved as wild-type mice. Therefore, endogenous kappa-receptor activation would tonically inhibit Ab response. Opioid receptor deficient mice were also used to investigate the immunosuppressive action of naltrindole, a delta-opioid receptor antagonist, shown earlier to inhibit graft rejection and the allogeneic mixed lymphocyte reaction (MLR) in vitro. Naltrindole and two related compounds inhibited MLR performed with lymphocytes from wild-type and delta-opioid receptor knockout mice. These compounds also suppressed MLR assayed with cells from triple mu/delta/kappa-opioid receptor mutants. We therefore demonstrate that naltrindole immunosuppressive activity is not mediated by any of the three mu-, delta- or kappa-opioid receptors, but by a target which remains to be discovered.

Autoradiographic mapping of the opioid receptor-like 1 (ORL1) receptor in the brains of mu-, delta- or kappa-opioid receptor knockout mice.

The opioid receptor-like 1 (ORL1) receptor shares a high degree of sequence homology with the classical mu-, delta- and kappa-opioid receptors and a functional mutual opposition between these receptors has been suggested. To further address this possible interaction we have used mu-, delta- and kappa-opioid receptor knockout mice to determine autoradiographically if there are any changes in the number or distribution of the ORL1 receptor, labelled with [(3)H]nociceptin, in the brains of mice deficient in each of the opioid receptors. An up-regulation of ORL1 expression was observed across all brain regions in delta-knockouts with cortical regions typically showing a 15-30% increase in binding that was most marked in heterozygous mice. In contrast, ORL1 receptor expression was down-regulated in virtually all brain structures in heterozygous kappa-knockouts although the magnitude of this change was not as great as for the delta-knockouts. No significant alterations in ORL1 receptor expression were observed across brain regions in mu-receptor knockout mice and there were no qualitative differences in ORL1 receptor expression in any groups. These data suggest there are interactions between the ORL1 system and the classical opioid receptors and that the interactions are receptor-specific. The greater differences observed in heterozygous mice suggest that these interactions might be most relevant when there is only partial loss of receptor function.

Detection of opioid receptor mRNA by RT-PCR reveals alternative splicing for the delta- and kappa-opioid receptors.

The three mu-, delta- and kappa-opioid receptors have recently been cloned and characterized at the molecular level. Our analysis of opioid receptor transcripts by RT-PCR revealed two PCR products derived from delta and kappa mRNAs with size higher than expected from the known cDNA sequences. DNA sequencing showed additional nucleotides inserted between the known splice sites, indicating the possible existence of alternative splicing pathways for delta and kappa receptors. The novel delta-opioid receptor transcript is expressed in mouse brain and contains a 243 bp insertion. This additional sequence is located at the splice junction between the first and second coding exons and is encoded by a single exon located 9 kb upstream exon 2 in the mDOR gene. The other alternative transcript occurs in human monocytic and T lymphocytic cell lines and encodes a novel form of the kappa-opioid receptor. The PCR product presents a 23 bp deletion at the 3' end of exon 2 followed by a 246 bp insertion found between exons 2 and 3. In the hKOR gene, this insertion is encoded by two DNA segments. One of them is located 0.4 kb downstream exon 2 while the second is flanking exon 3 on the 5' side. Both novel putative delta and kappa exons present in-frame stop codons that would lead to truncated receptor proteins. A possible functional or regulatory role of these shorter proteins in opioid function remains to be determined.

Quantitative autoradiography of mu-,delta- and kappa1 opioid receptors in kappa-opioid receptor knockout mice.

Mice deficient in the kappa-opioid receptor (KOR) gene have recently been developed by the technique of homologous recombination and shown to lack behavioural responses to the selective kappa1-receptor agonist U-50,488H. We have carried out quantitative autoradiography of mu-, delta- and kappa1 receptors in the brains of wild-type (+/+), heterozygous (+/-) and homozygous (-/-) KOR knockout mice to determine if there is any compensatory expression of mu- and delta-receptor subtypes in mutant animals. Adjacent coronal sections were cut from the brains of +/+, +/- and -/- mice for the determination of binding of [3H]CI-977, [3H]DAMGO (D-Ala2-MePhe4-Gly-ol5 enkephalin) or [3H]DELT-I (D-Ala2 deltorphin I) to kappa1-, mu- and delta-receptors, respectively. In +/- mice there was a decrease in [3H]CI-977 binding of approximately 50% whilst no kappa1-receptors could be detected in any brain region of homozygous animals confirming the successful disruption of the KOR gene. There were no major changes in the number or distribution of mu- or delta-receptors in any brain region of mutant mice. There were, however some non-cortical regions where a small up-regulation of delta-receptors was observed in contrast to an opposing down-regulation of delta-receptors evident in mu-knockout brains. This effect was most notable in the nucleus accumbens and the vertical limb of the diagonal band, and suggests there may be functional interactions between mu- and delta-receptors and kappa1- and delta-receptors in mouse brain.

Analysis of [3H]bremazocine binding in single and combinatorial opioid receptor knockout mice.

Despite ample pharmacological evidence for the existence of multiple mu-, delta- and kappa-opioid receptor subtypes, only three genes encoding mu-(MOR), delta-(DOR) and kappa-(KOR) opioid receptor have been cloned. The KOR gene encodes kappa(1)-sites, which specifically bind arylacetamide compounds, and the possible existence of kappa-opioid receptor subtypes derived from another kappa-opioid-receptor gene, yet to be characterized, remains a very contentious issue. kappa(2)-Opioid receptors are described as binding sites typically labelled by the non-selective benzomorphan ligand [3H]bremazocine in the presence of mu-, delta- and kappa(1)-opioid receptor blocking ligands. To investigate the genetic origin of kappa(2)-opioid receptors, we have carried out homogenate binding experiments with [3H]bremazocine in brains of single MOR-, DOR-, KOR- and double MOR/DOR-deficient mice. Scatchard analysis showed that 68+/-12% of the binding sites arise from the MOR gene, 27+/-1% from the DOR gene and 14.5+/-0.2% from the KOR gene, indicating that the three known genes account for total [3H]bremazocine binding. Experiments in the presence of mu-, delta- and kappa(1)-opioid receptor suppressor ligands further showed that non-kappa(1)-opioid receptor labelling can be accounted for by binding to both the mu- and delta-opioid receptors. Finally, [3H]bremazocine binding experiments performed on brain membranes from the triple MOR/DOR/KOR-deficient mice revealed a complete absence of binding sites, confirming definitively that no additional gene is required to explain the total population of [3H]bremazocine binding sites. Altogether the data show that the putative kappa(2)-opioid receptors are in fact a mixed population of KOR, DOR and predominantly MOR gene products.

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.

Involvement of neuropeptide FF receptors in neuroadaptive responses to acute and chronic opiate treatments.

BACKGROUND AND PURPOSE Opiates remain the most effective compounds for alleviating severe pain across a wide range of conditions. However, their use is associated with significant side effects. Neuropeptide FF (NPFF) receptors have been implicated in several opiate-induced neuroadaptive changes including the development of tolerance. In this study, we investigated the consequences of NPFF receptor blockade on acute and chronic stimulation of opioid receptors in mice by using RF9, a potent and selective antagonist of NPFF receptors that can be administered systemically. EXPERIMENTAL APPROACH The effects of RF9 were investigated on opioid pharmacological responses including locomotor activity, antinociception, opioid-induced hyperalgesia, rewarding properties and physical dependence. KEY RESULTS RF9 had no effect on morphine-induced horizontal hyperlocomotion and slightly attenuated the decrease induced in vertical activity. Furthermore, RF9 dose-dependently blocked the long-lasting hyperalgesia produced by either acute fentanyl or chronic morphine administration. RF9 also potentiated opiate early analgesic effects and prevented the development of morphine tolerance. Finally, RF9 increased morphine-induced conditioned place preference without producing any rewarding effect by itself and decreased naltrexone-precipitated withdrawal syndrome following chronic morphine treatment. CONCLUSION AND IMPLICATIONS The NPFF system is involved in the development of two major undesirable effects: tolerance and dependence, which are clinically associated with prolonged exposure to opiates. Our findings suggest that NPFF receptors are interesting therapeutic targets to improve the analgesic efficacy of opiates by limiting the development of tolerance, and for the treatment of opioid dependence.

Impaired striatum-dependent behavior in GASP-1-knock-out mice.

G protein-coupled receptor (GPCR) associated sorting protein-1 (GASP-1) is suspected to play a key role in recycling and degradation of several GPCRs. In a previous study, we have shown that GASP-1-knock-out (GASP-1-KO) mice displayed deficits in acquiring a cocaine self-administration task, associated with an exacerbated down-regulation of striatal dopaminergic and cholinergic receptors. Among several possibilities, GASP-1 deficiency could have impaired memory processes underlying the acquisition of the operant conditioning task. Therefore, the present study investigated cognitive performances of GASP-1-KO mice and their wild-type littermates (WT) in a broad variety of memory tasks. Consistent with a deficit in procedural memory, GASP-1-KO mice showed delayed acquisition of a food-reinforced bar-press task. During water-maze training in hidden- or visible-platform paradigms, mutant and WT mice acquired the tasks at the same rate. However, GASP-1 mice exhibited persistent thigmotaxic swimming, longer distance to the platform, and reduced swim speed. There was no deficit in several tasks requiring simple behavioral responses (Barnes maze, object recognition and passive avoidance tasks). Thus, the ability to acquire and/or express complex responses seems affected in GASP-1-deficient mice. Hippocampal functions were preserved, as the retention of an acquired memory in spatial tasks remained unaffected. The pattern of behavioral deficits observed in GASP-1-KO mice is coherent with current knowledge on the role of striatal GPCRs in acquisition/expression of skilled behavior and in motivation. Together with the previous findings, the so far established phenotype of GASP-1-KO mice makes them a potentially exciting tool to study striatal functions.

Identification of potential active-site residues in the human poly(ADP-ribose) polymerase.

The carboxyl-terminal catalytic domain of the human poly(ADP-ribose) polymerase (PARP) exhibits sequence homology with the NAD(P)(+)-dependent leucine and glutamate dehydrogenases. To clarify the role played by some conserved residues between PARP and NAD(P)(+)-dependent dehydrogenases, point mutations were introduced into the whole enzyme context. Non-conservative mutations of Lys-893 (K893I) and Asp-993 (D993A) completely inactivate human PARP, whereas conservative and nonconservative mutations of Asp-914 (D914E and D914A, respectively) and Lys-953 (K953R and K953I, respectively) partially alter PARP activity. The consequences of conservative substitution of Lys-893 and Asp-993 on the kinetic properties of human poly(ADP-ribose) polymerase enzyme and the polymer it synthesizes suggest that these 2 amino acids are directly involved in the covalent attachment of the first ADP-ribosyl residue from NAD+ onto the acceptor amino acid. In addition, the recent resolution of the three-dimensional structure of the NAD(+)-linked glutamate dehydrogenase from Clostridium symbiosum (Baker, P.J., Britton, K.L., Engel, P.C., Farrants, G.W., Lilley, K.S., Rice, D.W., and Stillman, T.J. (1992) Proteins 12, 75-86) strongly supports our alignment with leucine and glutamate dehydrogenases and provides an interesting structural framework for the analysis of our results of site-directed mutagenesis.

Detection of poly(ADP ribose) polymerase in crude extracts by activity-blot.

We have recently devised an activity-blot procedure permitting the detection, on the same nitrocellulose sheet, of the functional poly(ADP ribose) polymerase (PARP) activity as well as the immunostained active peptide(s) after renaturation of the transferred protein(s). Using this technique we have analyzed the PARP activity in higher and lower eukaryotes directly on crude extracts from cell cultures. This procedure has been extended also to in situ screening of bacterial colonies expressing the PARP enzymatic activity.

Characterization of delta, kappa, and mu human opioid receptors overexpressed in baculovirus-infected insect cells.

The cDNAs encoding human delta (hDOR), kappa (hKOR) and micro (hMOR) opioid receptors were cloned in the baculovirus Autographa californica (AcMNPV) under the control of the polyhedrin promoter with or without an amino-terminal hexahistidine tag. Expression levels were optimized in Spodoptera frugiperda (Sf9) cells and were in the following order hMOR > hDOR > hKOR. The receptors bound antagonists with affinity values similar to those published previously for the receptors expressed in mammalian cells. They also retained selectivity toward specific antagonists. The three receptors bound peptidic agonists with low affinity, suggesting that they might not be functionally coupled to intracellular effectors. Introduction of an amino-terminal hexahistidine tag decreased the levels of expression markedly. Only hMOR-his was expressed at a level allowing binding study, but no difference could be detected in the affinities of both agonists and antagonists compared with the nontagged protein. hMOR expression was also optimized in High Five cells leading to a further increase in protein production. The pharmacological profile was similar to the one obtained when the receptor was expressed in Sf9 cells. Our results show that the baculovirus expression system is suitable for large scale production of human opioid receptors.

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.

Poly(ADP-ribose)polymerase: a novel finger protein.

By Energy Dispersive X-ray fluorescence we have determined that calf thymus poly(ADP-ribose) polymerase binds two zinc ions per enzyme molecule. Using 65Zn (II) for detection of zinc binding proteins and polypeptides on western blots, we found that the zinc binding sites are localized in a 29 kd N-terminal fragment which is included in the DNA binding domain. Metal depletion and restoration experiments proved that zinc is essential for the binding of this fragment to DNA as tested by Southwestern assay. These results correlate with the existence of two putative zinc finger motifs present in the N-terminal part of the human enzyme. Poly(ADP-ribose)polymerase fingers could be involved in the recognition of DNA strand breaks and therefore in enzyme activation.