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O Civelli

Publications and source records attributed to O Civelli.

At least 37 records · Page 2Linked to original sources
PubMed1998

Adenosine A1 receptor-mediated modulation of dopamine D1 receptors in stably cotransfected fibroblast cells.

The antagonistic interactions between adenosine A1 and dopamine D1 receptors were studied in a mouse Ltk- cell line stably cotransfected with human adenosine A1 receptor and dopamine D1 receptor cDNAs. In membrane preparations, both the adenosine A1 receptor agonist N6-cyclopentyladenosine and the GTP analogue guanyl-5'-yl imidodiphospate induced a decrease in the proportion of dopamine D1 receptors in a high affinity state. In the cotransfected cells, the adenosine A1 agonist induced a concentration-dependent inhibition of dopamine-induced cAMP accumulation. Blockade of adenosine A1 receptor signal transduction with the adenosine A1 receptor antagonist 1,3-dipropyl-8-cyclopentylxanthine or with pertussis toxin pretreatment increased both basal and dopamine-stimulated cAMP levels, indicating the existence of tonic adenosine A1 receptor activation. Pretreatment with pertussis toxin also counteracted the effects of low concentrations of the A1 agonist on D1 receptor-agonist binding. The results suggest that adenosine A1 receptors antagonistically modulate dopamine D1 receptors at the level of receptor binding and the generation of second messengers.

Animals↗
PubMed1998

Expression and characterization of a dopamine D4R variant associated with delusional disorder.

Multiple genetic polymorphisms of the human dopamine D4 receptor (hD4R) have been identified including a 12 bp repeat in exon 1 associated with a psychotic condition called delusional disorder. Competition binding assays revealed minor pharmacological differences between the recombinant A1 (normal) and A2 (delusional) proteins with respect to quinpirole and the antipsychotic clozapine, however no functional differences were detected for receptor activation by dopamine, epinephrine, or norepinephrine. Our results suggest that this polymorphism may only confer susceptibility to delusional disorder in combination with other genetic or environmental factors.

Animals↗
PubMed1998

Structures that delineate orphanin FQ and dynorphin A pharmacological selectivities.

Strict pharmacological selectivity in families of structurally related ligands and receptors may result from a key process in evolution aiming at increasing diversity in neurotransmission. An intriguing example of such exclusive specificity can be found in the newly discovered orphanin FQ (OFQ) system when it is compared with the opioid system. Both OFQ and its receptor share a high degree of sequence similarity to the opioid peptides and their corresponding receptors, respectively. However, OFQ does not activate opioid receptors, nor do the opioid peptides elicit biological activity at the OFQ receptor. We have therefore investigated the basis for the inherent selectivity of the primary structures of OFQ and dynorphin A, its closest counterpart. A series of truncated and/or chimeric peptides led to the conclusion that both peptides contain domains which establish their pharmacological selectivity. In the OFQ molecule we could delineate a domain that prevents its ability to activate the kappa-opioid receptor by apparently repelling its binding. In both peptides the selectivity-generating domains are composed of single residues in key positions together with short stretches of amino acids which do not overlap. To prove this concept, we designed a universal agonist and found it active at both the OFQ receptor and the kappa-opioid receptor. Our observations suggest that a coordinated mechanism of evolution has separated the orphanin FQ system from the opioid system.

Amino Acid Sequence↗
PubMed1998

Development and characterization of antibodies directed against the mouse D4 dopamine receptor.

Polyclonal antibodies against the mouse D4 dopamine receptor have been developed in order to investigate the anatomical localization of this receptor in the mouse brain. Two antibodies were generated against specific peptides corresponding to predicted extracellular and intracellular regions of the D4 protein. Specificity of these antibodies was demonstrated on human embryonic kidney 293 (HEK 293) cells transfected with different dopamine receptor subtypes; immunoreactivity was detected only in cells transfected with the mouse D4 dopamine receptor cDNA. Following in vitro transcription/translation of the mouse D4 cDNA, a single protein band of 36 kDa was selectively immunoprecipitated with the anti-D4 antibodies. The antibodies also detected a single protein of 36 kDa in Western blot of HEK 293 cells transiently transfected with the mouse D4 receptor. These antibodies were able to detect the D4 receptor in several regions of the mouse brain. In the regions examined, D4 immunoreactivity was found in neurones located in layers II-VI of the frontal and piriform cortices, with the highest concentration in layer II; in scattered neurones in the caudate putamen and in larger neurones in the globus pallidus. In all experiments, both antibodies exhibit the same specificity, and all immunoreactivity could be abolished by preincubation with the corresponding peptide antigen.

Amino Acid Sequence↗
PubMed1998

Creating a functional opioid alkaloid binding site in the orphanin FQ receptor through site-directed mutagenesis.

Although much has been learned about the mechanisms of ligand selectivity between different opioid receptor subtypes, little is known about the common opioid binding pocket shared by all opioid receptors. The recently discovered orphanin system offers a good opportunity to study the mechanisms involved in the binding of opioid versus nonopioid ligands. In the current study, we adopt a "gain of function" approach aimed at shifting the binding profile of the orphanin FQ receptor toward that of the opioid receptors. After two rounds of mutagenesis, several orphanin FQ receptor mutants can be labeled with the opiate alkaloid [3H]naltrindole and show greatly increased affinities toward the opiate antagonists naltrexone, nor-binaltrophine HCl, and (-)-bremazocine. These orphanin FQ receptor mutants also display stereospecificity similar to that of opioid receptors. Furthermore, the orphanin FQ receptor mutant that has the best affinities toward the opioid alkaloids shows, in the presence of GTP and high salt concentration, an affinity-shift profile similar to that of the delta receptor. Most strikingly, the same mutant exhibits naltrindole-sensitive etorphine-stimulated [35S]guanosine-5'-O-(3-thio)triphosphate binding, whereas the effect of etorphine on GTP binding cannot be inhibited by naltrindole in the wild-type receptor. Our results indicate that 1) several residues in the orphanin FQ receptor are critical to its selectivity against the opiate alkaloids, particularly antagonists; and 2) mutating these residues to those of the opioid receptor at the corresponding position preserves the agonist/antagonist nature of opiate alkaloids as they interact with the mutant receptor. It is reasonable to hypothesize that the corresponding residues in the opioid receptors may form a functional common binding pocket for opiate alkaloids. These findings may be helpful to medicinal chemists in designing ligands for the orphanin FQ receptor based on the structure of the opiate alkaloids.

Amino Acid Substitution↗
PubMed1998

Reverse physiology: discovery of the novel neuropeptide, orphanin FQ/nociceptin.

The search for novel neurotransmitters and neuropeptides has been recently revolutionized by the development of a purification strategy based on orphan G protein-coupled receptors, cloned receptors for which no natural ligands are known. This strategy uses the orphan receptor as bait to identify its natural ligand. This article will review the discovery of the first natural ligand isolated following this strategy. This ligand is a peptide that shares some striking sequence similarity to the opioid peptides and has been named Orphanin FQ or Nociceptin (OFQ/NOC). The discovery of OFQ/NOC will be described as one example of the use of orphan receptors in identifying novel neurotransmitters and neuropeptides, an example that has already been followed in the identification of other novel neuropeptides. After reviewing the conceptual and technological basis of the strategy and its successful first application, we discuss the criteria used to validate OFQ/NOC as the natural ligand of the orphan receptor and as a genuine neuropeptide. We also discuss the importance and implications of discovering OFQ/NOC mode of synthesis, which is synthesized as expected in the form of a larger polypeptide precursor, which in turn raises the question of the existence of other OFQ/NOC-related peptides. We then present an overview of the numerous studies that have blossomed after the OFQ/NOC discovery and describe the numerous physiological roles that have already been attributed to OFQ/NOC, and in particular the controversy regarding its involvement in pain perception. Because of the similarities between the OFQ/NOC and opioid systems, we also discuss overlaps between these systems and present evidence favoring a pharmacological separation between these systems. We finish by outlining the power of the orphan receptor strategy and by discussing some of its pitfalls.

Animals↗
PubMed1997

Orphanin FQ acts as an anxiolytic to attenuate behavioral responses to stress.

Orphanin FQ (OFQ, Nociceptin) is a recently discovered 17-amino acid neuropeptide that is structurally related to the opioid peptides but does not bind opioid receptors. OFQ has been proposed to act as an anti-opioid peptide, but its widespread sites of action in the brain suggest that it may have more general functions. Here we show that OFQ plays an important role in higher brain functions because it can act as an anxiolytic to attenuate the behavioral inhibition of animals acutely exposed to stressful/anxiogenic environmental conditions. OFQ anxiolytic-like effects were consistent across several behavioral paradigms generating different types of anxiety states in animals (light-dark preference, elevated plus-maze, exploratory behavior of an unfamiliar environment, pharmacological anxiogenesis, operant conflict) and were observed at low nonsedating doses (0.1-3 nmol, intracerebroventricular). Like conventional anxiolytics, OFQ interfered with regular sensorimotor function at high doses (>3 nmol). Our results show that an important role of OFQ is to act as an endogenous regulator of acute anxiety responses. OFQ, probably in concert with other major neuropeptides, exerts a modulatory role on the central integration of stressful stimuli and, thereby, may modulate anxiety states generated by acute stress.

Animals↗
PubMed1997

Epinephrine and norepinephrine act as potent agonists at the recombinant human dopamine D4 receptor.

The catecholamines dopamine (DA), epinephrine (EP), and norepinephrine (NE) play important roles in learning and memory, emotional states, and control of voluntary movement, as well as cardiovascular and kidney function. They activate distinct but overlapping neuronal pathways through five distinct DA receptors (D1R-D5R) and at least 10 different adrenergic receptors (alpha 1a/b/c, alpha 2a/b/c-1/c-2, and beta 1/beta 2/beta 3). The D4R, which is localized to mesolimbic areas of the brain implicated in affective and emotional behavior, has a deduced amino acid sequence with homology to both adrenergic and dopaminergic receptor subtypes. We report here that DA, EP, and NE all show binding in the nanomolar range to three isoforms of the recombinant human D4R (hD4R): D4.2, D4.4, and D4.7. Submicromolar concentrations of DA, EP, and NE were sufficient to activate hD4R isoforms in two different functional assays: agonist-induced guanosine 5'-O-(3-[35S]thiotriphosphate) binding and modulation of adenylyl cyclase activity. DA was approximately fivefold more potent than EP and NE at the D4R, whereas activation of the human D2R required at least 100-fold higher catecholamine concentrations. Functional activation of the D4R by multiple neurotransmitters may provide a novel mechanism for integration of catecholamine signaling in the brain and periphery.

Adrenergic alpha-Agonists↗
PubMed1997

Interaction of [3H]orphanin FQ and 125I-Tyr14-orphanin FQ with the orphanin FQ receptor: kinetics and modulation by cations and guanine nucleotides.

The heptadecapeptide orphanin FQ (OFQ) has been identified as the endogenous ligand for a G protein-coupled receptor (OFQ-R), which, despite its high degree of sequence similarity to opioid receptors, fails to bind opioid ligands. We developed two radioligands for the OFQ-R: a tritiated native OFQ peptide ([3H]OFQ) and a radioiodinated form in which Leu14 was substituted by tyrosine (125I-Tyr14-OFQ). Their binding properties were examined in human embryonic kidney (HEK) 293 and Chinese hamster ovary (CHO) cells heterologously expressing the OFQ-R at different levels (HEK 293 expressed 40-fold more OFQ-R than did CHO). Both ligands exhibited rapid, monophasic association kinetics in each cell line. Dissociation of both ligands from OFQ-R expressed in HEK 293 cells was biphasic, whereas dissociation of 125I-Tyr14-OFQ from OFQ-R expressed in CHO cells was monophasic and slow. Saturation binding analysis revealed two affinity states in HEK 293 cells with binding parameters in accord with those determined kinetically. In CHO cells, 125I-Tyr14-OFQ detected a single affinity state with an intermediate Kd value of 54 pM. Optimal binding of the radioligands required 1-5 mM MgCl2, whereas millimolar concentrations of ZnCl2, CaCl2, MnCl2, and NaCl reduced specific binding of both ligands. A nonhydrolyzable GTP analog [guanosine-5'-(beta,gamma-imido)triphosphate] reduced the affinity of both OFQ ligands to their receptor without significant changes in the total binding capacity, indicating functional interactions between the OFQ-R and G proteins. In rat brain membranes, specific, saturable binding of 125I-Tyr14-OFQ was demonstrated to be pharmacologically identical to the heterologously expressed OFQ-R. Taken together, these results indicate that 125I-Tyr14-OFQ and [3H]OFQ exhibit virtually identical characteristics and are suitable for the pharmacological analysis of the OFQ-R.

Animals↗
PubMed1996

Moving from the orphanin FQ receptor to an opioid receptor using four point mutations.

It is unclear how receptor/ligand families that are evolutionarily closely related achieve functional separation. To address this question, we focus here on the newly discovered Orphanin FQ, a peptide homologous to the opioid peptide Dynorphin, and its receptor, the Orphanin FQ receptor, which is highly homologous to the opioid receptors. In spite of this high degree of homology in terms of both ligands and receptors, there is little direct cross-talk between the Orphanin FQ system and the endogenous opioid system. Thus, the opioid peptides show either relatively low affinity or no affinity toward the Orphanin FQ receptor; conversely, Orphanin FQ has no affinity toward any of the opioid receptors. We sought to investigate the molecular basis of such discrimination by attempting to reverse it and endowing the Orphanin FQ receptor with the ability to bind opioids. We report that by mutating as few as four amino acids, we can produce a receptor that recognizes pro-Dynorphin products with very high affinity and yet still binds Orphanin FQ as well as the wild-type receptor. This suggests that the Orphanin FQ receptor has developed features that specifically exclude the opioids and that these features are distinct from those required for the high affinity binding of its own endogenous ligand.

Analgesics↗
PubMed1996

Modulation of voltage-gated calcium channels by orphanin FQ in freshly dissociated hippocampal neurons.

Orphanin FQ (OFQ) has recently been reported to be an endogenous ligand for the opioid-like LC132 receptor. The effect of OFQ on high voltage-gated calcium channels (VGCCs) was examined in freshly dissociated rat pyramidal neurons using the whole-cell configuration of the patch-clamp technique. High-threshold Ba2+ currents were reversibly inhibited by OFQ. The depression of the currents was associated with a slowed rate of activation and a change in the activation I-V relationship at step potentials higher than +30 mV. In concentration-response experiments, a mean (+/-SEM) pEC50 value of 7.0 +/- 0.07 and a Hill coefficient of 1.5 +/- 0.08 (n = 5) were obtained. The near-maximum inhibition of the Ba2+ currents by OFQ (1 microM) amounted to 31 +/- 2.2% of control (n = 15). Opioid receptors could not account for the effects of OFQ on VGCCs, because naloxone, a broad spectrum mu-, delta-, and kappa-receptor antagonist, did not reduce the effectiveness of OFQ. When GTP-gamma-S was included in the pipette, the depression of the currents by OFQ was irreversible, whereas currents from neurons preincubated with pertussis toxin were not inhibited by OFQ, consistent with the involvement of a PTX-sensitive G-protein. When selective blockers of VGCCs were used, it was demonstrated that all subtypes of VGCCs were affected by OFQ. In conclusion, the effect of OFQ on VGCCs expressed in hippocampal CA3 and CA1 neurons may play an important role in the regulation of hippocampal cell excitability and neurotransmitter release.

Animals↗
PubMed1996

Primary structure and tissue distribution of the orphanin FQ precursor.

The heptadecapeptide orphanin FQ (OFQ) is a recently discovered neuropeptide that exhibits structural features reminiscent of the opioid peptides and that is an endogenous ligand to a G protein-coupled receptor sequentially related to the opioid receptors. We have cloned both the human and rat cDNAs encoding the OFQ precursor proteins, to investigate whether the sequence relationships existing between the opioid and OFQ systems are also found at the polypeptide precursor level, in particular whether the OFQ precursor would encode several bioactive peptides as do the opioid precursors, and to study the regional distribution of OFQ sites of synthesis. The entire precursor protein displays structural homology to the opioid peptide precursors, especially preprodynorphin and preproenkephalin. The predicted amino acid sequence of the OFQ precursor contains a putative signal peptide and one copy of the OFQ sequence flanked by pairs of basic amino acid residues. Carboxyl-terminal to the OFQ sequence, the human and rat precursors contain a stretch of 28 amino acids that is 100% conserved and thus may encode novel bioactive peptides. Two peptides derived from this stretch were synthesized but were found to be unable to activate the OFQ receptor, suggesting that if they are produced in vivo, these peptides would likely recognize receptors different from the OFQ receptor. To begin analyzing the sites of OFQ mRNA synthesis, Northern analysis of human and rat tissues were carried out and showed that the OFQ precursor mRNA is mainly expressed in the brain. In situ hybridization of rat brain slices demonstrated a regional distribution pattern of the OFQ precursor mRNA, which is distinct from that of the opioid peptide precursors. These data confirm that the OFQ system differs from the opioid system at the molecular level, although the OFQ and opioid precursors may have arisen from a common ancestral gene.

Amino Acid Sequence↗
PubMed1996

The novel neuropeptide orphanin FQ fails to produce conditioned place preference or aversion.

An unbiased conditioned place preference procedure was used to determine whether the newly-identified neuropeptide orphanin FQ produced motivational effects after intracerebroventricular microinjections. Microinjections of orphanin FQ (0.1-100 nmol) failed to produce conditioned place preference or aversion, but a pronounced motor impairment was observed during conditioning sessions with the two highest doses. Thus, it appears that orphanin FQ lacks motivational effects when administered at behaviourally active doses.

Animals↗
PubMed1996

Structure-activity relationship studies on the novel neuropeptide orphanin FQ.

The heptadecapeptide orphanin FQ (OFQ) is an endogenous ligand to an opioid-like G protein-coupled receptor. Although the primary structure of OFQ exhibits some similarity to the opioid peptides, OFQ is not recognized by opioid receptors nor does the OFQ receptor bind opioid ligands. In order to investigate the structural determinants of this ligand/receptor selectivity, we conducted a systematic structure-activity study on OFQ to characterize which sites of the molecule are important for receptor activation. Alanine- and D-amino acid-scanning mutagenesis revealed several residues in the amino-terminal half of OFQ which participate in both receptor binding and activation. Most strikingly, the Phe1 position could be changed to a tyrosine without loss of biological activity. In addition, the OFQ receptor seemed to require recognition of the complete peptide molecule for activation. These results indicate that the mode of interaction of OFQ with its receptor may be different from that of the opioid peptides with their respective receptors and might therefore account for the observed selectivity.

Amino Acid Sequence↗
PubMed1996

Rats rapidly develop tolerance to the locomotor-inhibiting effects of the novel neuropeptide orphanin FQ.

We examined the effects of intracerebroventricular (i.c.v.) administration of orphanin FQ (OFQ) on locomotor activity in rats. The rats were habituated to locomotor-testing boxes and then injected i.c.v. with OFQ (0 - 10 nmoles). Acute injections of OFQ produced dose-orderly reductions in horizontal locomotion and rearing activity. This suppression of motor activity was characterized by a disruption of balance and muscle control. Within minutes of i.c.v. injection of the higher doses of OFQ, the rats exhibited flaccid muscle tone. They each lay in an atypical posture, pressing the abdomen against the floor, and splaying the hindlimbs. When these rats locomoted, their gate was unsteady. They wobbled from side to side, and frequently fell over. Repeated daily injections of OFQ resulted in a rapid development of tolerance to the OFQ-induced suppression of locomotion and rearing activity. Tolerance to the observed impairments of motor control were also apparent. In the rats that were repeatedly treated with the highest dose (10 nmol) of OFQ, tolerance to the motoric effects was still apparent after 7 days without OFQ treatment.

Analysis of Variance↗
PubMed1996

Orphanin FQ is a functional anti-opioid peptide.

The heptadecapeptide orphanin FQ has recently been shown to be the endogenous agonist for the orphan opioid-like receptor, LC132. The molecular evidence that LC132 and orphanin FQ are evolutionarily related to other opioid receptors and their ligands suggests that these proteins may also play a role in modulating opiate actions. We now report that orphanin FQ (0.5-10 nmol), injected intracerebroventricularly in mice, does not produce hyperalgesia as suggested previously but rather reverses opioid-mediated (i.e. naloxone-sensitive) stress-induced antinociception in three different algesiometric assays. In addition to its antagonism of endogenous opioid antinociception, orphanin FQ dose-dependently (2.5-25 nmol) reverses systemic morphine antinociception (5 mg/kg, s.c.). Based on these data, we propose that orphanin FQ is a functional anti-opioid peptide.

Acetates↗
PubMed1996

Molecular attributes of dopamine receptors: new potential for antipsychotic drug development.

The neurotransmitter dopamine (DA) plays a central role in the control of motor function, emotional states, and endocrine physiology. The discovery that schizophrenic symptoms can be alleviated by neuroleptic drugs and the finding that these drugs interact at dopamine receptors has indicated involvement of the dopamine system in schizophrenia. The dopamine system has recently been shown to rely on the activation of five distinct subtypes of DA receptors (D1-D5) identified by molecular cloning, and pharmacological studies have specifically implicated the D2-like receptors (D2R, D3R and D4R) in antipsychotic action. In addition, the localization of D3R and D4R expression in the mesolimbic/mesocortical DA pathways is consistent with their proposed involvement in affective behaviour, and suggests that drugs developed specifically for these receptor subtypes might have potent antipsychotic activity with a lower propensity for extrapyramidal, endocrine, and cognitive side-effects.

Antipsychotic Agents↗