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Objectives The recent finding that gastroenteropancreatic neuroendocrine tumors expressed the dopaminergic D2 receptor in addition to somatostatin (sst) receptors suggested that multiple targeting approaches might decrease hormone hypersecretion more effectively than sst agonists alone. Methods To test this hypothesis, (i) we measured the expression of sst receptor type 2 (sst2 receptor) and D2 receptor in 11 gastroenteropancreatic neuroendocrine tumors and (ii) we compared the ability of lanreotide, cabergoline, their combination, and sst/D2 chimeric ligands to decrease chromogranin A (CgA), gastrin, or serotonin release in primary cultures derived from these tumors. Results Moderate to high positivity was observed for sst2 receptor and D2 receptor, the latter being more expressed in pancreatic tumors. Lanreotide decreased CgA secretion in all cultures, but only 3 tumors responded to cabergoline. No additivity was observed in lanreotide. BIM 23A781 decreased CgA release to the same extent as lanreotide, whereas the other chimeric ligands were less efficient. However, BIM 23A781 was 50 times less potent than lanreotide. Similar patterns were found for gastrin or serotonin. Conclusion No improvement was brought by the sst/D2 combination or chimeric ligands. Factors that underlie these tissue-specific differences remain to be elucidated.

In the sequence of the C-terminal octapeptide of cholecystokinin, the phenylalanine amide residue in position 33 is of primary importance for the biological activity. Indeed, removal of Phe33-NH2 is a modification known to induce antagonist properties. The influence of the chemical nature of the Phe33-NH2 side chain on the biological activity of CCK8 was investigated through replacement of this residue by several amino acids with different lipophilic properties in the sequence of Boc(Nle28, Nle31)CCK27-33, an equipotent analogue of CCK8. The binding properties of these new CCK-related analogues: Boc(Nle28,Nle31,X33)CCK27-33 were measured on both mouse brain and guinea pig pancreatic membranes, and their peripheral activities on amylase secretion and contractions of guinea pig ileum. Among the various peptides modified in position 33, Boc(Nle28,Nle31,Naa33-NH2)CCK27-33(Naa = naphthylalanine) and Boc(Nle28,Nle31,Cha33-NH2)CCK27-33(Cha = cyclohexylalanine) displayed high affinities for central and peripheral CCK-receptors and proved to be full agonists of CCK8 in the peripheral tests while Boc(Nle28,Nle31,Ala33-NH2)CCK27-33 was completely inactive. This suggests that, at the level of the Phe33-NH2 subsite, the critical factor for optimal interaction with CCK-receptors is not the aromatic nature of the side chain but its size and hydrophobicity.

F-FDG) have greatly improved the diag-nosis and staging of numerous tumors with somatostatin-receptor-subtype overexpression or active glucose metabo-lism,respectively.Thisentailedthedevelopmentofnumerousradiolabeled peptides that target other receptors overex-pressed by tumors that do not accumulate somatostatinanalogues or

In this work, we evidenced characteristic features of agonist-induced trafficking of receptor stimulus for the rat neurotensin receptor 1 (NTS1). Thus, reverse potency orders between two agonists, EISAI-1 and neuromedin N, were observed in inositol 1,4,5-trisphosphate and cAMP assays in Chinese hamster ovary cells transfected with this receptor. Indeed, compared with other agonists, EISAI-1 presented lower relative potency toward inositol 1,4,5-trisphosphate production than toward cAMP accumulation, guanosine 5'-O -(3-[35 S]thio)triphosphate binding, and [3H]arachidonic acid production. These results indicated pathway-dependent differences in EISAI-1 intrinsic efficacies, favoring activations of Gs- and Gi/o-related pathways over the Gq/11-related pathway. Moreover, although coupling to Gq/11 and Gi/o involved the third intracellular loop and the C-terminal domain of the NTS1 receptor, respectively, we demonstrated that deletion of the latter domain suppressed agonist-induced cAMP accumulation, suggesting that this domain also mediated coupling to Gs. Together, these results indicated that, unlike other agonists, EISAI-1 discriminated between the pathways involving the receptor C-terminal domain and that involving the third intracellular loop. These properties of EISAI-1 were also observed in cortical neurons endogenously expressing the NTS1 receptor. They were further attributed to the functionalization of its COOH end by an ethyl group, because the unesterified analog EISAI-2 presented normal behavior on inositol 1,4,5-trisphosphate production. These findings support the hypothesis of agonist-selective receptor states with distinct conformations or accessibilities of intracellular domains. They also suggest that the differential involvement of these domains in coupling to G proteins might represent a molecular basis for agonist-selective responses through G protein-coupled receptors.

Recent studies demonstrated that the chemokine monocyte chemoattractant protein-1 (MCP-1)/CCL2 and its receptor, CCR2, play important roles in various brain diseases. In this study, using quantitative autoradiography, we studied the pharmacological properties of [125l]MCP-1/CCL2 binding in rat brain and we clearly showed the distribution of CCR2 receptors in cerebral cortex, nucleus accumbens, striatum, amygdala, thalamus, hypothalamus, hippocampus, substantia nigra, mammillary bodies and raphe nuclei. Moreover, using double fluorescent immunohistochemistry, we showed that CCR2 receptors were constitutively expressed on neurons and astrocytes. Using RT-PCR methods, we demonstrated that CCR2 mRNA is present in various brain areas described above. Four hours after an acute intraperitoneal lipopolysaccharide injection, we showed that MCP-1/CCL2 binding was up-regulated in several brain structures; this effect took place on both CCR2B labelled neurons and astrocytes and to a lesser extent on activated microglia. To explore neurobiological function of CCR2, actimetric study was carried out. After intracerebroventricular injections of MCP-1/CCL2, we showed that motor activity was markedly decreased. Our results provide the first evidence for constitutive CCR2 receptor expression with precise neuroanatomical and cellular localizations in the brain, and its regulation during an inflammatory process, suggesting that MCP-1/CCL2 and CCR2 play important physiological and pathophysiological role(s) in the CNS.

Recent findings suggest that gonadal steroid hormones are neuroprotective and may provide clinical benefits in delaying the development of Parkinson's disease. In this report we investigated the ability of oestradiol to protect mesencephalic dopaminergic neurones cultured in serum-free or serum-supplemented medium from toxicity induced by 6-hydroxydopamine or 1-methyl-4-phenylpyridinium ion (MPP+). The efficiency of both toxins and oestradiol was evaluated by tyrosine hydroxylase (TH) immunocytochemistry, [3H]dopamine ([3H]DA) uptake, length of dopaminergic processes and lactate dehydrogenase (LDH) release measurement. In cultures grown in serum-supplemented medium, a 2-h pre-treatment with high concentrations (10-100 microM) of 17beta-oestradiol or 17alpha-oestradiol, the stereoisomer with weak oestrogenic activity, protected both dopaminergic and non-dopaminergic neurones from toxicity induced by 6-hydroxydopamine (6-OHDA; 40 or 100 microM) and by the high MPP+ concentrations (50 microM) necessary to obtain significant neuronal death under those culture conditions. At these concentrations, MPP+ was no longer selective for dopaminergic neurones but affected all cells present in the culture. In contrast, the hormonal treatments did not protect against selective degeneration of dopaminergic neurones induced by lower MPP+ concentrations (below 10 microM), related to inhibition of complex I of respiratory chain. In cultures grown in serum-free medium, oestradiol concentrations higher than 1 microM induced neuronal degeneration and no protection against 6-OHDA or MPP+ toxicity was observed at lower concentrations of the steroid. The neuroprotective effects of 17alpha- or 17beta-oestradiol evidenced in this model might be due to the antioxidant properties of these compounds. However, other non-genomic effects of the steroids cannot be excluded.

Neurotensin is present in selective mesolimbic dopaminergic projections to the nucleus accumbens (NAc) shell but also is synthesized locally in this region and in the motor-associated NAc core. We examined the electron microscopic immunolabeling of the high-affinity neurotensin receptor (NTR) and neurotensin in these subdivisions of rat NAc to determine the sites for receptor activation and potential regional differences in distribution. Throughout the NAc, NTR immunoreactivity was localized discretely within both neurons and glia. NTR-labeled neuronal profiles were mainly axons and axon terminals with diverse synaptic structures, which resembled dopaminergic and glutamatergic afferents, as well as collaterals of inhibitory projection neurons. These terminals had a significantly higher numerical density in the NAc core than in the shell but were prevalent in both regions, suggesting involvement in both motor and limbic functions. In each region, neurotensin was detected in a few NTR-immunoreactive axon terminals and in terminals that formed symmetric, inhibitory type synapses with NTR-labeled somata and dendrites. The NTR labeling, however, was not seen within these synapses and, instead, was localized to segments of dendritic and glial plasma membranes often near excitatory type synapses. Neuronal NTR immunoreactivity also was associated with cytoplasmic tubulovesicles and nuclear membranes. Our results suggests that, in the NAc shell and core, NTR is targeted mainly to presynaptic sites, playing a role in the regulated secretion and/or retrograde signaling in diverse, neurotransmitter-specific neurons. The findings also support a volume mode of neurotensin actions, specifically affecting excitatory transmission through activation of not only axonal but also dendritic and glial NTR. J. Comp. Neurol. 435:142–155, 2001. © 2001 Wiley-Liss, Inc.

Previous work from our laboratory showed prevention of 1-methyl-4-phenyl-1,2,3,6 tetrahydropyridine (MPTP) induced dopamine depletion in striatum of C57Bl/6 mice by 17β-estradiol, progesterone, and raloxifene, whereas 17α-estradiol had no effect. The present study investigated the mechanism by which these compounds exert their neuroprotective activity. The hormonal effect on the dopamine transporter (DAT) was examined to probe the integrity of dopamine neurons and glutamate receptors in order to find a possible excitotoxic mechanism. Drugs were injected daily for 5 days before MPTP (four injections, 15 mg/kg ip at 2-h intervals) and drug treatment continued for 5 more days. MPTP induced a decrease of striatal DAT-specific binding (50% of control) and DAT mRNA in the substantia nigra (20% of control), suggesting that loss of neuronal nerve terminals was more extensive than cell bodies. This MPTP-induced decrease of striatal [125I]RTI-121 specific binding was prevented by 17β-estradiol (2 μg/day), progesterone (2 μg/day), or raloxifene (5 mg/kg/day) but not by 17α-estradiol (2 μg/day) or raloxifene (1 mg/kg/day). No treatment completely reversed the decreased levels of DAT mRNA in the substantia nigra. Striatal [125I]RTI-121 specific binding was positively correlated with dopamine concentrations in intact, saline, or hormone-treated MPTP mice. Striatal NMDA-sensitive [3H]glutamate or [3H]AMPA specific binding remained unchanged in intact, saline, or hormone-treated MPTP mice, suggesting the unlikely implication of changes of glutamate receptors in an excitotoxic mechanism. These results show a stereospecific neuroprotection by 17β-estradiol of MPTP neurotoxicity, which is also observed with progesterone or raloxifene treatment. The present paradigm modeled early DA nerve cell damage and was responsive to hormones. Synapse 41:131–138, 2001. © 2001 Wiley-Liss, Inc.

The aim of the present study was to investigate the role of neurotensin in the regulation of NT1 receptors during postnatal development in the rat brain. Characterization of the ontogeny of neurotensin concentration and [125I]neurotensin binding to NT1 receptors in the brain at different embryonic and postnatal stages showed that neurotensin was highly expressed at birth, reaching peak levels at postnatal day 5 (P5) and decreasing thereafter. The transient rise in neurotensin levels preceded the maximal expression of NT1 receptors, observed at P10, suggesting that neurotensin may influence the developmental profile of NT1 receptors. Using primary cultures of cerebral cortex neurons from fetal rats, we showed that exposure to the neurotensin agonist JMV 449 (1 nM) decreased (−43%) the amount of NT1 receptor mRNA measured by reverse transcription-PCR, an effect that was abolished by the nonpeptide NT1 receptor antagonist SR 48692 (1 μM). However, daily injection of SR 48692 to rat pups from birth for 5, 9, or 15 days did not modify [125I]neurotensin binding in brain membrane homogenates. Moreover, postnatal blockade of neurotensin transmission did not alter the density and distribution of NT1 receptors assessed by quantitative autoradiography nor NT1 receptor mRNA expression measured by in situ hybridization in the cerebral cortex, caudate-putamen, and midbrain. These results suggest that although NT1 receptor expression can be regulated in vitro by the agonist at an early developmental stage, neurotensin is not a major factor in the establishment of the ontogenetic pattern of NT receptors in the rat brain. J. Neurosci. Res. 60:362–369, 2000 © 2000 Wiley-Liss, Inc.

Earlier studies have demonstrated overexpression of NT1 neurotensin receptors in rat brain during the first 2 weeks of life. To gain insight into this phenomenon, we investigated the identity and distribution of NT1 receptor proteins in the brain of 10-day-old rats by using two different NT1 antibodies: one (Abi3) directed against the third intracellular loop and the other (Abi4) against the C-terminus of the receptor. Immunoblot experiments that used Abi3 revealed the presence of two differentially glycosylated forms of the NT1 receptor in developing rat brain: one migrating at 54 and the other at 52 kDa. Whereas the 54-kDa form was expressed from birth to adulthood, the 52-kDa form was detected only at 10 and 15 days postnatal. Only the 52-kDa isoform was recognized by Abi4. By immunohistochemistry, both forms of the receptor were found to be predominantly expressed in cerebral cortex and dorsal hippocampus, in keeping with earlier radioligand binding and in situ hybridization data. However, whereas Abi4 immunoreactivity was mainly concentrated within nerve cell bodies and extensively colocalized with the Golgi marker alpha-mannosidase II, Abi3 immunoreactivity was predominantly located along neuronal processes. These results suggest that the transitorily expressed 52-kDa protein corresponds to an immature, incompletely glycosylated and largely intracellular form of the NT1 receptor and that the 54-kDa protein corresponds to a mature, fully glycosylated, and largely membrane-associated form. They also indicate that antibodies directed against different sequences of G-protein-coupled receptors may yield isoform-specific immunohistochemical labeling patterns in mammalian brain. Finally, the selective expression of the short form of the NT1 receptor early in development suggests that it may play a specific role in the establishment of neuronal circuitry.

The postnatal ontogeny of the levocabastine-sensitive neurotensin receptor (NT2) mRNA was studied by in situ hybridization in the rat brain and compared with the distribution of the levocabastine-insensitive NT1 receptor. NT2 receptor mRNA was absent at birth from all brain structures except the ependymal cell layer lining the ventricles. The development of NT2 receptor mRNA followed three ontogenetic patterns. The first pattern, involving the majority of the cerebral gray matter, was characterized by a continuous increase from postnatal day 5 (P5) to P30. The second one, involving regions rich in myelinated fibers such as the corpus callosum and lacunosum moleculare layer of the hippocampus, exhibited a pronounced increase between P5 and P10, peaked at P15 and was followed by a plateau or a slight decrease. The third pattern was observed in the ependymal cell layer lining the olfactory and lateral ventricles, where the high labeling already present at birth continued to increase during development. These different developmental patterns could reflect the variety of cells expressing NT2 receptor mRNA, including neurons, protoplasmic astrocytes in gray matter, fibrous astrocytes present in myelinated fibers tracts, and ependymal cells. In contrast, NT1 receptor mRNA, which seems to be associated only with neurons, was highly and transiently expressed during the perinatal period in the cerebral cortex, hippocampus and striatal neuroepithelium. Other regions, notably the ventral tegmental area and substantia nigra compacta, exhibited a gradual increase in NT1 receptor signal, reaching adult levels by P21. Both the differential localization and ontogenetic profiles of NT1 and NT2 receptor mRNAs suggest different involvement of these two receptors in brain functions and development.

Opiates and cannabinoids are among the most widely consumed habit-forming drugs in humans. Several studies have demonstrated the existence of interactions between both kind of drugs in a variety of effects and experimental models. The present study has been focused to determine whether perinatal delta9-tetrahydrocannabinol (Delta9-THC) exposure affects the susceptibility to reinforcing effects of morphine in adulthood and whether these potential changes were accompanied by variations in mu opioid receptor binding in brain regions related to drug reinforcement. Adult female rats born from mothers that were daily treated with delta9-THC during gestation and lactation periods, exhibited a statistically significant increase in the rate of acquisition of intravenous morphine self-administration behavior when compared with females born from vehicle-exposed mothers, an effect that did not exist in delta9-THC-exposed male offspring. This increase was significantly greater on the last day of acquisition period. There were not significant differences when the subjects were lever pressing for food. In parallel, we have also examined the density of mu opioid receptors in the brain of adult male and female offspring that were exposed to Delta9-THC during the perinatal period. Collectively, perinatal exposure to delta9-THC produced changes in mu opioid receptor binding that differed regionally and that were mostly different as a function of sex. Thus, delta9-THC-exposed males exhibited a lower density for these receptors than their respective oil-exposed controls in the caudate-putamen area as well as in the amygdala (posteromedial cortical nucleus). On the contrary, delta9-THC-exposed females exhibited higher density of these receptors than their respective oil-exposed controls in the prefrontal cortex, the hippocampus (CA3 area), the amygdala (posteromedial cortical nucleus), the ventral tegmental area and the periaqueductal grey matter, whereas the binding was lower than control females only in the lateral amygdala. These results support the notion that perinatal delta9-THC exposure alters the susceptibility to morphine reinforcing effects in adult female offspring, in parallel with changes in mu opioid receptor binding in several brain regions.

The present study describes the characterization of the binding properties and autoradiographic distribution of a new nonpeptide antagonist of neurotensin receptors, [3H]SR 142948A (2-[[5-(2,6-dimethoxyphenyl)-1-(4-(N-(3-dimethylaminopropyl)-N-methyl carbamoyl)-2-isopropylphenyl)-1H-pyrazole-3-carbonyl]-amino]-ad amantane-2-carboxylic acid, hydrochloride), in the rat brain. The binding of [3H]SR 142948A in brain membrane homogenates was specific, time-dependent, reversible and saturable. [3H]SR 142948A bound to an apparently homogeneous population of sites, with a Kd of 3.5 nM and a Bmax value of 508 fmol/mg of protein, which was 80% higher than that observed in saturation experiments with [3H]neurotensin. [3H]SR 142948A binding was inhibited by SR 142948A, the related nonpeptide receptor antagonist, SR 48692 (2-[[1-(7-chloroquinolin-4-yl)-5-(2,6-dimethoxyphenyl)-1H-pyrazole -3-carbonyl]amino]-adamantane-2-carboxylic acid) and neurotensin. Saturation and competition studies in the presence or absence of the histamine H1 receptor antagonist, levocabastine, revealed that [3H]SR 142948A bound with similar affinities to both the levocabastine-insensitive neurotensin NT1 receptors (20% of the total binding population) and the recently cloned levocabastine-sensitive neurotensin NT2 receptors (80% of the receptors) (Kd = 6.8 and 4.8 nM, respectively). The regional distribution of [3H]SR 142948A binding in the rat brain closely matched the distribution of [125I]neurotensin binding. In conclusion, these findings indicate that [3H]SR 142948A is a new potent antagonist radioligand which recognizes with high affinity both neurotensin NT1 and NT2 receptors and represents thus an excellent tool to study neurotensin receptors in the rat brain.

This study demonstrates the adhesion and growth of bovine corneal epithelial cells on the surface of a new hydrogel. The hydrogel, containing 78% of water and 22% of AN-69 polymer (poly(acrylonitrile-sodium methallyle sulfonate)), was obtained by phase inversion of polymer–dimethylformamide solution in physiological saline (0.9% NaCl). Experiments were also carried out using hydrogel treated with arginine, human albumin and collagen IV. Covering of hydrogel samples by epithelial cells was completed within ten days, with good cell viability. The epithelial cells spread out and formed a consistent cell layer, confirmed by immunocytochemistry experiments against cytokeratins. Transmission electron micrographs showed numerous desmosomes between cells and the presence of some membrane differentiations at the cell/hydrogel interface. This study suggests therefore that the hydrogel might be suitable for the development of artificial epikeratoplasty grafts.

Neurotensin has been suggested to be involved in neurological and mental disorders associated with altered dopaminergic transmission. The lack of a potent neurotensin receptor antagonist had prevented us from studying the real physiological implication of this peptide in brain function. We thus recently developed such a non-peptide neurotensin receptor antagonist, SR 48692, (2-(1-(7-chloroquinolin-4-yl)-5-(2,6-dimethoxyphenyl)-1H-pyrazole- 3-carbonyl)amino)-adamantane-2-carboxylic acid), which appeared to be potent in various central and peripheral preparations. In the present study, we tested the pharmacological properties of SR 48692 and of two optically synthetic analogs of this compound on neurotensin binding to both adult guinea-pig brain membrane homogenates and coronal brain sections, as well as on neurotensin stimulation of the K(+)-evoked release of [3H]dopamine in guinea-pig striatal slices. Our results demonstrated that (1) high-affinity neurotensin binding sites are present in the guinea-pig brain in regions rich in both dopamine cell bodies and terminals; (2) the binding of neurotensin is inhibited by SR 48692 and its related S(+) active analog, SR 48527, with IC50 values in the nM range and (3) the non-peptide antagonist has no agonist effect but antagonizes neurotensin-induced [3H]dopamine release from guinea-pig striatal nerve terminals.

Many studies have reported the presence of high amounts of neurotensin (NT) binding sites in the mesencephalon of adult rat, and their possible role in mediating the effects of the peptide on the activity of mesencephalic dopaminergic neurons. In the present study, we demonstrate the presence of NT sites in primary cultures of embryonic rat mesencephalic cells. On these cells, a single class of high affinity 125 I-NT binding sites was observed. The value of the apparent affinity constant (0.3 nM) did not show any significant change throughout time, from 3 to 14 days in culture. The number of sites, however, increased until day 11 and decreased thereafter. Acetylneurotensin (8–13), NT and neuromedin N were potent competitors of 125 I-NT binding, while NT (1–10), NT (1–11) and levocabastine were uneffective. These results indicate that the sites detected in the mesencephalic cultures share common binding properties with the high-affinity NT sites already described in adult rat brain. The neuronal localization of the NT sites was suggested by their presence in neuron-enriched serum-free cultures and their absence in glial cultures. Autoradiographic studies confirmed the cellular localization of NT sites and indicated that, under our experimental conditions, cells labeled by 125 I-NT represented 0.14% of the initially plated cell number. Taken together, these results show that the development of mesencephalic neurons in primary culture is associated with an increased expression of NT binding sites. Since such cultures have been shown previously to contain functional dopaminergic neurons, we suggest that they could provide a good model to investigate the modulation of the activity of these neurons by NT.

Binding properties and localization of [3H]ohmefentanyl, a new ligand for mu opioid receptors, were investigated on normal human brain sections. Binding assays performed at the level of the basal ganglia revealed: (1) a steady-state binding reached after 60 min incubation at room temperature, (2) the presence, in saturation experiments, of an apparent single class of binding sites with a Kd = 1.68 +/- 0.45 nM and a Bmax = 162 +/- 9 fmol/mg protein, (3) an order of potency to inhibit [3H]ohmefentanyl binding as follows: ohmefentanyl greater than [D-Ala2, MePhe4, Gly-ol5] enkephalin (DAGO) greater than ethylketocyclazocine (EKC) much greater than Tyr-D-Ser(OtBu)-Gly-Phe-Leu-Thr(OtBu) (BUBU) and U-50,488H. Quantitative autoradiography showed an heterogeneous distribution of [3H]ohmefentanyl binding sites with the highest densities in amygdala, medical geniculate body, thalamus, and caudate nucleus. Binding characteristics and anatomical distribution also show that [3H]ohmefentanyl may bind to a small proportion of additional sites called "DAGO-inaccessible [3H]ohmefentanyl specific binding sites." [3H]Ohmefentanyl binding to these sites can be partly inhibited by sigma ligands such as 1,3-di-o-tolylguanidine (DTG) and haloperidol. However, unlabeled DAGO inhibited more than 80% of [3H]ohmefentanyl specific binding in most of the human brain regions studied, suggesting that the major population of sites labeled by [3H]ohmefentanyl represented mu opioid receptors.

[ 3 H]Dihydrotetrabenzine ([ 3 H]TBZOH), a derivative of the monoamine depleting agent, tetrabenazine, has been shown to bind to the vesicular monoamine transporter. We studied the effect of unilateral nigral lesion with 6-hydroxydopamine on [ 3 H]TBZOH binding in the striatum by means of quantitative autoradiography. Topographical analysis of striatal [ 3 H]TBZOH binding showed in control rats a pattern similar to the known distribution of dopaminergic innervation, and allowed to visualize dopaminergic denervation in lesioned rats. A good correlation was found between striatal [ 3 H]TBZOH binding levels and tyrosine hydroxylase activities in similar areas on adjacent slices following 6-OHDA lesions with varying severity. These data reveal that autoradiography of [ 3 H]TBZOH binding is a good index for the visualization and quantitation of dopaminergic nerve terminals integrity in the striatum.

High-affinity binding sites for neurotensin 1–13 are found transiently in the human inferior olive during development. An in vitro membrane binding assay demonstrates, in the fetus, a specific binding to a single population of sites ( K d = 561pM) which are l levocabastine-insensitive and possess a B max value of 71 fmol/mg protein. No specific binding is present in the adult. Film radioautography shows, in fetuses of 15 weeks, a labelling in the whole inferior olives. At 25 weeks and at 33 weeks, the labelling is concentrated in the ventral lamella of the principal olive and in the caudal accessory olives. At birth and until 1 month of age, the labelling is still detected in the medial part of the ventral lamella of the principal olive. The labelling disappears after 3 months of age, is absent at 4 and 6 months and remains absent in the adults from 55 to 81 years of age. At the light microscopic level, the labelling is located mostly outside olivary neuronal cell bodies suggesting its presence on afferent nerve fibers. This ontogeny and localization of the binding sites in the inferior olive is quite different from that of the neurotensin 1–13 nerve terminals.

International audience; Chronic use of psychostimulants induces enduringly increased responsiveness to a subsequent psychostimulant injection and sensitivity to drug-associated cues, contributing to drug craving and relapse. Neurotensin (NT), a neuropeptide functionally linked to dopaminergic neurons, was suggested to participate in these phenomena. We and others have reported that SR 48692, an NT receptor antagonist, given in pre- or co-treatments with cocaine or amphetamine, alters some behavioral effects of these drugs in rats. However, its efficacy when applied following repeated cocaine administration remains unknown. We, therefore, evaluated the ability of SR 48692, administered after a cocaine regimen, to interfere with the expression of locomotor sensitization and conditioned place preference (CPP) in rats. We demonstrated that the expression of locomotor sensitization, induced by four cocaine injections (15 mg/kg, i.p.) every other day and a cocaine challenge 1 week later, was attenuated by a subsequent 2-week daily administration of SR 48692 (1 mg/kg, i.p.). Furthermore, the expression of cocaine-induced CPP was suppressed by a 10-day SR 48692 treatment started after the conditioning period (four 15 mg/kg cocaine injections every other day). Taken together, our data show that a chronic SR 48692 treatment given after a cocaine regimen partly reverses the expression of locomotor sensitization and CPP in the rat, suggesting that NT participates in the maintenance of these behaviors. Our results support the hypothesis that targeting neuromodulatory systems, such as the NT systems may offer new strategies in the treatment of drug addiction.

International audience; Three neurotensin (NT) receptors have been cloned to date, two of which, NTS1 and NTS2, belong to the family of seven transmembrane domain receptors coupled to G proteins (GPCRs). NTS1 and NTS2 may activate multiple signal transduction pathways, involving several G proteins. However, whereas NT acts as an agonist towards all NTS1-mediated pathways, this peptide may exert either agonist or antagonist activities, depending on the NTS2-mediated pathway in question. Studies on these receptors reinforce the concept of independence between multiple signals potentially mediated through a single GPCR, generating a wide diversity of functional responses depending on the host cell and the ligand.

The cyclo-oxygenase (COX) and lipoxygenase (LOX) pathways belong to the eicosanoid synthesis pathway, a major component of the chronic inflammatory process occurring in Alzheimer's disease (AD). Clinical studies reported beneficial effects of COX inhibitors, but little is known about the involvement of LOXs in AD pathogenesis. beta-amyloid peptide (A beta) accumulation contributes to neurodegeneration in AD, but mechanisms underlying A beta toxicity have not been fully elucidated yet. Here, using an antisense oligonucleotide-based strategy, we show that blockade of 12-LOX expression prevents both A beta-induced apoptosis and overexpression of c-Jun, a factor required for the apoptotic process, in cortical neurons. Conversely, the 12-LOX metabolite, 12(S)-HETE (12(S)-hydroxy-(5Z, 8Z, 10E, 14Z)-eicosatetraenoic acid), promoted c-Jun-dependent apoptosis. Specificity of the 12-LOX involvement was further supported by the observed lack of contribution of 5-LOX in this process. These data indicate that blockade of 12-LOX expression disrupts a c-Jun-dependent apoptosis pathway, and suggest that 12-LOX may represent a new target for the treatment of AD.

The dopamine D2 receptor (D2R) in the nucleus accumbens (NAc) shell is implicated in schizophrenia and in psychostimulant-induced drug-seeking behavior, both of which are affected by activation of the functionally opposed high-affinity neurotensin receptor (NTS1). To determine the functionally relevant sites, we examined the dual electron microscopic immunocytochemical localization of D2R and NTS1 in the NAc shell of rat brain. Immunolabeling for each receptor was seen in association with cytoplasmic organelles, or more rarely, on the plasma membrane of both axonal and somatodendritic profiles. Some of the axonal and many of the dendritic processes colocalized the two receptors. The dually labeled axon terminals often formed symmetric synapses or appositional contacts with unlabeled dendritic profiles. The morphology of these terminals suggests that they contain either inhibitory amino acids or dopamine. Other axonal profiles expressing exclusively NTS1 or D2R were without synaptic specializations or formed asymmetric, excitatory-type synapses mainly on unlabeled dendritic spines. In addition, however, several D2R-immunoreactive terminals were observed presynaptic to dendrites containing NTS1. The somatodendritic profiles immunolabeled for NTS1 and/or D2R had morphological features typical of inhibitory spiny projection neurons in the NAc. These results suggest that activation of NTS1 and D2R can dually modulate transmitter release from the same or separate phenotypically distinct axon terminals in the NAc shell. These presynaptic receptors as well as the postsynaptic NTS1 distribution in neurons that also contain or receive input from terminals containing D2R may mediate the opposing actions of neurotensin and dopamine in the NAc.

This unit describes procedures for performing competition binding assays with neurotensin receptor subtypes 1 and 2 (NTS1 and NTS2). Binding assays using cloned receptors, brain membranes, and primary cultured mesencephalic neurons are presented. NTS1 binding assays employing either radiolabeled neurotensin or SR 48692 (a nonpeptide neurotensin antagonist) as radioligands are described. These procedures may be used to screen selective ligands at neurotensin receptor subtypes. Keywords: neurotensin; SR 48692; neurotensin receptors; NTS1; NTS2; binding assay; brain membranes; mesencephalic neurons

SUMMARY Neurotensin (NT) exerts multiple functions in the central nervous system and peripheral tissues. Its actions are mainly mediated by a high-affinity G-protein-coupled receptor, the NT-1 receptor. In this study we demonstrated a nuclear NT binding site in different cellular models. We first noted that a large percentage of NT-1 receptor cell body immunoreactivity was located in the nuclear soma and nuclear envelope of rat substantia nigra, a brain area rich in NT-containing axon terminals. The NT-1 receptor was also visualized in purified nuclei from CHO cells stably transfected with NT-1 receptor coupled to the enhanced green fluorescence protein by immunocytochemistry. We observed that both the nuclear envelope and the nuclear soma were labeled, and the labeling intensity significantly increased after NT agonist treatment. These results suggested that NT-1 receptors, present in both the nuclear soma and the nuclear envelope, can be modulated by the ligand. Lastly, [ 125 I]-NT binding experiments performed on isolated nuclei from a human lung cancer cell line endogenously expressing NT-1 receptor and NT, LNM35, revealed the existence of nuclear Gpp(NHp)-sensitive binding sites. These binding sites markedly decreased when cells were chronically treated with an NT-1 receptor antagonist, SR 48692. Taken together, these data suggest that the agonist regulates the expression of nuclear NT-1 receptors. (J Histochem Cytochem 52:335‐345, 2004)

Neurotensinergic cells were visualized by immunocytochemistry in rat hypothalamic cultures grown in serum-free medium for 12 days. They represented 0.03% of the total cell population. The pattern observed for the evolution of neurotensin content in hypothalamic cultures, from day 5 to day 21 (7-fold increase), was similar to that observed in the rat hypothalamus during the corresponding period of in vivo ontogeny, from birth to 19 days postnatal (6-fold increase).

Accumulation of amyloid beta peptide (Abeta) has been suggested to contribute to neurodegeneration in Alzheimer's disease (AD). Since chronic inflammation occurs in AD pathogenesis and lipoxygenases are important mediators of inflammatory processes, we evaluated the effect of lipoxygenase inhibitors on apoptosis induced by Abeta on rat cortical cells. The 12-lipoxygenase inhibitor baicalein attenuated both neuronal apoptosis and c-jun protein over-expression induced by Abeta(25- 35), whereas no protection was found with the broad spectrum lipoxygenase inhibitor nordihydroguaiaretic acid or the 5-lipoxygenase inhibitor caffeic acid. These results suggest that 12-lipoxygenase participates in a c-jun-dependent apoptosis pathway triggered by Abeta(25-35), and that specific 12-lipoxygenase inhibitors might be of interest in AD.

Neurotensin (NT) produces various stimulatory effects on dopaminergic neurons of the rat substantia nigra. To gain insight into the subcellular substrate for these effects, we compared by electron microscopy the distribution of immunoreactive high-affinity NT receptor proteins (NTRH) with that of high-affinity125I-NT binding sites in this region of rat brain. Quantitative analysis showed a predominant association of immunogold and radioautographic labels with somata and dendrites of presumptive dopaminergic neurons, and a more modest localization in myelinated and unmyelinated axons and astrocytic leaflets. The distributions of immunoreactive NTRH and125I-NT binding sites along somatodendritic plasma membranes were highly correlated and homogeneous, suggesting that membrane-targeted NTRH proteins were functional and predominantly extrasynaptic. Abundant immunocytochemically and radioautographically labeled receptors were also detected inside perikarya and dendrites. Within perikarya, these were found in comparable proportions over membranes of smooth endoplasmic reticulum and Golgi apparatus, suggesting that newly synthesized receptor proteins already possess the molecular and conformational properties required for effective ligand binding. By contrast, dendrites showed a proportionally higher concentration of immunolabeled than radiolabeled intracellular receptors. A fraction of these immunoreactive receptors were found in endosomes, suggesting that they had undergone ligand-induced internalization and were under a molecular conformation and/or in a physical location that precluded their recognition by and/or access to exogenous ligand. Our results provide the first evidence that electron microscopic immunocytochemistry of the NT receptor identifies sites for both the binding and trafficking of NT in the substantia nigra.

International audience; The present experiments were designed to assess the role of endogenous neurotensin (NT) in the behavioral response to acute and daily cocaine, after administration of the NT receptor antagonist, SR 48692. Given that glucocorticoids increase the sensitivity to the psychomotor effects of drugs of abuse, we also investigated the effects of SR 48692 on basal and cocaine-induced corticosterone secretion. Acute administration of SR 48692 (1 mg/kg i.p.) reduced the number of rearings induced by cocaine (15 mg/kg i.p.), without modifying horizontal activity. Repeated pretreatment with SR 48692 (1 mg/kg x 5 days) markedly reduced locomotion and rearings after an acute cocaine challenge (day 1), whereas the lower dose of SR 48692 (0.1 mg/kg) had no effect. SR 48692 (1 mg/kg), given daily before cocaine, also decreased cocaine-induced rearing on day 2, but had no effect on the following drug challenges (days 3-10). One week after discontinuing repeated cocaine injections, SR 48692 blocked vertical, but not horizontal, activity induced by an acute cocaine challenge. Rats treated repeatedly with cocaine showed an enhanced behavioral response characterized by the development of stereotypes, which were unaffected by SR 48692. Finally, treatment with SR 48692 did not alter corticosterone circadian secretion nor cocaine-stimulated corticosterone levels, indicating that the attenuation of the behavioral effects of cocaine after NT receptor blockade is not associated with blunted glucocorticoid secretion. These results indicate that administration of SR 48692 attenuates the locomotion and rearing response to cocaine but fails to modify stereotyped behavior, suggesting that SR 48692 modulates the behavioral effects of psychostimulant drugs by acting selectively on the mesolimbic dopaminergic system.

In spite of numerous hodological and neuropsychological studies emphasizing the multimodal connections and integrative functions of the retrosplenial cortex in primates, the precise fate of its caudoventral extent and the composition of the merging area with the hippocampal formation remain a matter of debate. We reported previously how the anlage of the retrosplenial cortex merges with the immature presubicular zone in the fetal rhesus monkey at the end of the first trimester of gestation. In the present study, this caudal area was further defined on a chemoarchitectonic basis, particularly during the late prenatal and perinatal stages, which correspond to the development of the cingulate sulcus and temporal gyri, and the differentiation of the retrosplenial/subicular complex. Neurotensin (NT), a pyramidal cell marker in the limbic cortex, and parvalbumin (PV), a marker of a subset of inhibitory local circuit neurons in the hippocampal formation, were used as immunocytochemical markers. According to distinct chemoarchitectural patterns, (1) areas 29l and 29m of the retrosplenial cortex formed a triangle-shaped ventral expansion which merged with a similar but dorsal expansion of the pre/parasubicular fields. A temporal extension of area 29m down to area TH could not be detected. The pre/parasubiculum contributed with area 29m to the lateral bank of the calcarine sulcus as far as the most caudal extent of the hippocampal formation. (2) The lamina principalis interna of the presubiculum was well individualized and did not appear as a simple horizontal shift of adjoining fields. (3) NT and PV displayed a distinct temporal profile of development. NT was already expressed in the pyramidal cells of the prospective retrosplenial cortex and ventral hippocampal formation at E47 (term 165 days). Major pathways of the hippocampal formation and retrosplenial cortex (fimbria, fornix, angular and cingulum bundles) were progressively labeled indicating early developing projections. A large set of NT-positive afferents reached the retrosplenial cortex between E114 and E120. Their laminar distribution was compatible with a thalamic or a subicular origin. (4) The development of PV expression was delayed until the last quarter of gestation, supporting its proposal as a signal of functional onset. The developmental fate and the particular connections of the presubiculum suggest that its functional importance should be further investigated during infancy and adulthood.

Receptors for the neuropeptide, neurotensin, were localized by immunohistochemistry in the rat brain by using an antibody raised against a sequence of the third intracellular loop of the cloned high affinity receptor. Selective receptor immunostaining was observed throughout the brain and brainstem. This immunostaining was totally prevented by preadsorbing the antibody with the immunogenic peptide. The regional distribution of the immunoreactivity conformed for the most part to that of [3H]- or [125I]-neurotensin binding sites previously identified by autoradiography. Thus, the highest levels of immunostaining were observed in the islands of Calleja, diagonal band of Broca, magnocellular preoptic nucleus, pre- and parasubiculum, suprachiasmatic nucleus, anterodorsal nucleus of the thalamus, substantia nigra, ventral tegmental area, pontine nuclei and dorsal motor nucleus of the vagus, all of which had previously been documented to contain high densities of neurotensin binding sites. There were, however, a number of regions reportedly endowed with neurotensin binding sites, including the central amygdaloid nucleus, periaqueductal gray, outer layer of the superior colliculus and dorsal tegmental nucleus, which showed no or divergent patterns of immunostaining, suggesting that they might be expressing a molecularly distinct form of the receptor. At the cellular level, neurotensin receptor immunoreactivity was predominantly associated with perikarya and dendrites in some regions (e.g., in the basal forebrain, ventral midbrain, pons and rostral medulla) and with axons and axon terminals in others (e.g., in the lateral septum, bed nucleus of the stria terminalis, neostriatum, paraventricular nucleus of the thalamus and nucleus of the solitary tract). These data indicate that neurotensin may act both post- and presynaptically in the central nervous system and confirm that some of its effects are exerted on projection neurons. There were also areas, such as the cerebral cortex, nucleus accumbens and para- and periventricular nucleus of the hypothalamus, which contained both immunoreactive perikarya/dendrites and axon terminals, consistent with either a joint association of the receptor with afferent and efferent elements or its presence on interneurons. Taken together, these results also suggest that the neurotensin high affinity receptor protein is associated with a neuronal population that is more extensive than originally surmised from in situ hybridization studies.

The rat neurotensin receptor was expressed in Spodoptera frugiperda insect (Sf9) cells using infection with a recombinant baculovirus. Immunoblot experiments performed with an antibody raised against the C-terminus of the receptor showed major bands at 47 (corresponding to the unglycosylated receptor protein) and 50 kDa, and minor bands at 65 and 36 kDa. The expressed receptor bound 125I-neurotensin with high affinity, was coupled to endogenous G-proteins, and agonist-induced inositol phosphate production was observed at early times after infection. These results show that the rat neurotensin receptor retains functional properties when expressed in the heterologous insect cell system.

International audience; Neurotensin (NT) has been shown to be involved in neuroendocrine regulation, and the presence of both the peptide and its receptors has been demonstrated in the hypothalamus. In the present study, we show that hypothalamic neurons in primary cultures express the neurotensin receptor (NTR) and we examined a possible regulation of this receptor by glucocorticoids and activators of adenylate cyclase. In the hypothalamic cultures, 125I-NT bound to a single class of binding sites, presenting a selectivity similar to that observed for the high-affinity NTR previously described in the adult rat brain. Radioautographic studies demonstrated that these 125I-NT binding sites were present on 3% of the neurons. A 48-h treatment with forskolin (fsk) decreased 125I-NT binding by 30%. No effect of dexamethasone (dex) alone was found on that parameter. However, a combined treatment with both agents led to a 40% decrease in 125I-NT binding, corresponding to a reduced number of binding sites, and to a 68% decrease in the amount of NTR mRNA. In parallel, the dex plus forsk treatment increased NT release in the incubation medium. Moreover, the decreases in 125I-NT binding and NTR mRNA induced by this treatment were abolished in the presence of an anti-NT antibody or SR 48692, a non-peptidic antagonist of NTR, suggesting that the down-regulation of NTR observed after dex plus fsk treatment was mediated by the release of endogenous NT. Agonist-induced down-regulation of the NTR in this system was confirmed by the application of an exogenous NT analogue, JMV 449. The present findings indicate that, in hypothalamic cultures, dex and fsk indirectly down-regulate NTR expression via the release of endogenous NT.

Wound healing is the main cause of the failure of filtering surgery in glaucoma. We developed a liposomal delivery system of mitoxantrone (MITX), an anthracyclin derivative, to allow a single adjuvant administration and to lessen ocular side-effects of the drug. In order to evaluate the antiproliferative activity of liposomal MITX, an ex vivo model consisting in the culture of subconjunctival tissue explants from rabbits pretreated with subconjunctival injections of free or liposomal MITX was used. We found that both forms of MITX decreased the growth rate as well as the explant proliferation surfaces 15 days or 1 month after a single administration of the drug in vivo. A morphometric analysis of the cells showed that the surface of the fibroblasts exposed to both forms of MITX was from 10 to 12 times as important as that of the control cells exposed to the empty liposomes and to the control buffer. A radioautographic study showed that more than 95% of the fibroblasts exposed to both forms of MITX were in the G1 phase of the cell cycle, while the control cell population was equally distributed among the different phases of the cell cycle.

In the present study, the effects of glucocorticoids and forskolin, an activator of adenylate cyclase, were examined on neurotensin (NT) production from rat hypothalamic neurons in primary culture. Treatment with dexamethasone induced a dose-dependent increase in NT content. The maximum was reached at 1 microM dexamethasone, which induced a 100% increase in NT levels. The effect of dexamethasone was mimicked by the glucocorticoid agonist RU28362 and blocked by the antiglucocorticoid RU38486, suggesting that this effect was mediated through the glucocorticoid receptor. The treatment with dexamethasone also enhanced the number of immunoreactive NTergic cells (92% increase). In contrast to dexamethasone, forskolin affected neither the NT content nor the number of immunoreactive NTergic cells. However, when cells were treated with both dexamethasone and forskolin, a 285% increase in NT content and a 430% increase in the number of immunoreactive NTergic cells were observed, representing 2.8- and 4.7-fold increases, respectively, compared to the effect of dexamethasone alone. Moreover, this combined treatment increased the accumulation of NT in the culture medium (160% increase) as well as the abundance of NT messenger RNA. We conclude from the present findings that dexamethasone and forskolin act synergistically to enhance NT production in hypothalamic neurons.

In this work, the molecular forms of the rat neurotensin receptor (NTR) expressed in transfected Chinese hamster ovary (CHO) cells, in infected Sf9 insect cells and in rat cerebral cortex were immunologically detected by means of an anti-peptide antibody raised against a fragment of the third intracellular loop of the receptor. Immunoblot experiments against a fusion protein indicated that the anti-peptide antibody recognized, under denaturing conditions, the corresponding amino acid sequence within the NTR. In immunoblot analysis of membranes from NTR-transfected CHO cells, high levels of immunoreactivity were observed between 60 and 72 kDa, while only a faint labelling was observed at 47 kDa, the molecular mass deduced for the rat NTR cDNA. The bands of high molecular mass were no longer observed after deglycosylation of membrane proteins by peptide N-glycosidase F, indicating that they represented glycosylated forms of the receptor. Extracts of membranes derived from baculovirus-infected Sf9 insect-cells expressing the NTR provided a quite different immunoblot pattern, since the major band detected in that case was at 47 kDa, the molecular size of the non-glycosylated receptor. Taken together, these data show that, while most of the NTR protein was glycosylated in CHO cells, it was unglycosylated in Sf9 insect-cells. In addition, molecular sizes of the receptor proteins observed in these two cell lines differed from those obtained for the NTR endogenously expressed in the rat cerebral cortex of 7 day-old rats, where bands at 56 and 54 kDa were detected. Binding experiments carried out on membrane preparations obtained from baculovirus-infected Sf9 cells demonstrated that the immunogenic sequence was still accessible to the antibody when the receptor was embedded in the cell membrane. Immunohistochemical studies carried out on both transfected CHO cells and infected Sf9 cells confirmed this interpretation and further indicated that the antibody could be applied in the visualization of the receptor.

In rat striatal slices, the increase (114 +/- 11%) in K(+)-evoked [3H]dopamine release induced by neurotensin (10 nM) was antagonized by 2-[(1-(7-chloro-4-quinolinyl)-5-(2,6-dimethoxyphenyl)pyrazol-3-yl) carboxylamino]tricyclo(3.3.1.1.3.7)decan-2-carboxylic acid (SR 48692, IC50 = 1.2 +/- 0.11 nM). SR 48692 (100 nM) also suppressed the neurotensin (10 nM)-induced increase (47%) in K(+)-evoked [3H]dopamine release in primary cultures of fetal rat mesencephalic cells. These results further characterize SR 48692 as a potent antagonist of neurotensin receptors in the rat.

Vasoactive intestinal peptide (VIP), a 28-amino acid peptide, plays a multifunctional neuromodulatory role in both peripheral and central nervous systems. We have recently reported that VIP induces interferon (IFN) alpha/beta synthesis in human colon adenocarcinoma cell line HT-29. It has been reported that VIP may counteract HIV-induced neuronal cell death; therefore, we postulated that the action of VIP may be mediated by a cascade regulation, involving the production of some cytokines such as IFN. Here we demonstrate that primary cultures of rat mesencephalic neurons and glial cells respond differently to VIP. Thus VIP enhanced 2'5' oligoadenylate (2'5' A) synthetase activity and inhibited vesicular stomatitis virus multiplication in glial cultures only. However, both cell cultures had functional adenylate cyclase coupled receptors for VIP. The increase in 2'5'A synthetase activity in glial cultures reached a maximum with 10(-6) M VIP and required cellular RNA and protein synthesis. Anti-IFN alpha/beta, but not anti-IFN gamma, antibodies abolished the induction of the antiviral and 2'5'A synthetase activities by VIP in rat glial-enriched cultures, suggesting that these inductions were mediated through IFN alpha/beta synthesis. Moreover, VIP or poly (i). poly (C12U) caused, in the glial cultures, the induction and secretion of an IFN of type alpha/beta with a titer value of 16 and 32 units/ml respectively. In contrast, neither of these two substances was able to induce IFN synthesis in neurons, which were, however, sensitive to IFN alpha/beta produced by VIP-treated glial cells. IFN produced by VIP in glial cells may therefore play an important role in defending the brain against viruses.

The cellular localization of the cocaine binding sites in primary cultures of embryonic rat mesencephalic cells was previously reported to differ from that observed in adult rat brain. In order to know whether this different localization was associated with a different pharmacological selectivity, we tested the effect of new cocaine analogs on tritiated dopamine ([3H]DA) uptake in primary cultures of rat embryonic mesencephalic cells. In these cultures, [3H]DA was taken up by a nomifensine-sensitive, but desipramine and fluoxetine-insensitive process, reflecting selective uptake by the dopaminergic transporter. 3 beta-(4-Chlorophenyl)tropan-2 beta-carboxylic acid methyl ester (RTI-COC-31) was by far the most potent inhibitor of the [3H]DA uptake, presenting an IC50 of 3.8 nM, while the corresponding analog with an unsubstituted phenyl ring (WIN 35,065-2) was 38 times less potent. The enantiomer of WIN 35,065-2, namely WIN 35,065-3, was 30 times less potent than the former. A similar pattern was found for the relative ability of these compounds to inhibit binding of the radiolabeled cocaine derivative [125I]RTI-55 to membranes prepared from mesencephalic cultures. The order of potencies found for the three cocaine analogs on mesencephalic cultures was similar to that previously obtained in [3H] WIN 35,428 binding experiments and [3H]DA uptake inhibition in adult rat striatum, suggesting that the pharmacological selectivity of cocaine sites functionally related to the DA transporter in cultured embryonic neurons does not differ from that obtained in adult rat brain.

We have recently demonstrated that [3H]ohmefentanyl, a non-peptidergic opioid ligand which was suggested to cross the blood brain barrier in contrast to other peptidergic opioid ligands, bound not only to mu opioid receptor sites but also to sigma sites. In order to examine whether [3H]ohmefentanyl can be used as a marker for mu sites, we investigated the effects of brain lesions on [3H]ohmefentanyl binding site densities, as compared with [3H][D-Ala2, MePhe4, Gly-ol5]enkephalin ([3H]DAGO), a selective mu ligand. These binding site densities were measured by quantitative autoradiography in the rat striatum and substantia nigra, two brain structures known to contain a high density of mu receptors, following lesions of the nigro-striatal dopaminergic pathway and striatal intrinsic neurons. Following unilateral nigral lesion with 6-hydroxydopamine, [3H]ohmefentanyl binding site densities were decreased in the patches (-35%) and matrix (-20%) of the ipsilateral striatum and in the lesioned substantia nigra pars compacta (-49%). Unilateral striatal lesion with quinolinic acid induced 72%, 61% and 50% decreases in [3H]ohmefentanyl binding in the patches and matrix of the lesioned striatum and in the ipsilateral substantia nigra pars reticulata, respectively. Similar results were obtained in the binding of [3H]DAGO. Indeed, a significant linear correlation was observed between [3H]ohmefentanyl and [3H]DAGO binding site densities. Therefore, mu opioid receptors may be mainly located on intrinsic neurons in the striatum, dopaminergic cell bodies in the substantia nigra pars compacta and nerve terminals of striatal efferents in the substantia nigra pars reticulata.(ABSTRACT TRUNCATED AT 250 WORDS)

Ohmefentanyl has been shown to be 6300 times more potent than morphine for analgesia. The receptor binding characteristics and distribution of [3H]ohmefentanyl in rat brain sections are presented. [3H]Ohmefentanyl bound with high affinity to opioid receptors in a saturable manner (Kd = 0.95 +/- 0.08 nM, Bmax = 337 +/- 14 fmol/mg protein). We used various currently available specific mu, delta and kappa ligands to show that [3H]ohmefentanyl has a high selectivity for the mu opioid receptor. However, [D-Ala2,MePhe4,Gly-ol5]enkephalin (DAGO) was unable to completely inhibit [3H]ohmefentanyl specific binding, while complete inhibition was observed with fentanyl derivatives and the benzomorphan derivative, ethylketocyclazocine. This remaining 20% DAGO-inaccessible [3H]ohmefentanyl specific binding did not correspond to either mu1, delta or kappa sites. Haloperidol and 1,3-di-o-tolylguanidine were able to inhibit DAGO-inaccessible [3H]ohmefentanyl specific binding, suggesting that [3H]ohmefentanyl might also bind to haloperidol-sensitive sigma sites. The topographical distribution of [3H]ohmefentanyl found by autoradiography was generally similar to that of [3H]DAGO. However, in agreement with the biochemical results, quantitative analysis revealed additional sites in several rat brain regions, the greatest discrepancies with [3H]DAGO distribution being observed in cerebellum, central grey, hippocampal formation and locus coeruleus. Finally, our results suggest that this capacity of binding to both mu and sigma sites is shared by various fentanyl derivatives.

Recently, we reported bilateral increases in striatal neurotensin (NT) levels following unilateral 6-hydroxydopamine lesion of the nigrostriatal dopaminergic pathway. In the present study, the effect of unilateral striatal lesions with quinolinic acid (QA, 300 nmol) or ibotenic acid (IBO, 130 nmol) on striatal NT levels and binding site densities were analyzed in order to investigate other possible regulations of NT systems. QA and IBO injection decreased γ-aminobutyric acid (GABA) levels and [125I]iodosulpride (a specific D2 receptor antagonist) binding site densities in the lesioned striatum, indicating degeneration of striatal intrinsic neurons. Striatal dopaminergic terminals were not altered by QA as shown by the lack of changes in [3H]dihydrotetrabenazine ([3H]TBZOH, a specific ligand of the vesicular monoamine transporter) binding site densities. Moreover, QA lesion induced an increase in NT levels and a decrease in NT binding sites in the lesioned striatum without any change in the contralateral structure. In contrast to QA, IBO might destroy a certain proportion of dopaminergic terminals in the lesioned striatum, as shown by a 54% decrease in [3H]TBZOH binding. Furthermore, IBO lesion enhanced striatal NT levels bilaterally, while NT binding sites decreased in the lesioned striatum and increased in the contralateral side. The present results suggest that not only dopaminergic neurons but also striatal intrinsic neurons may control NT systems in the striatum.

Dermorphin, Tyr-DAla-Phe-Gly-Tyr-Pro-Ser-NH2, a potent opioid peptide isolated from amphibian skin, is endowed with outstanding structural and biological features. It has no common structure with mammalian opioid peptides and is a unique example of a peptide, synthesized by an animal cell, which contains a D-amino acid in its native sequence. We have undertaken a complete evaluation of the receptor selectivity of dermorphin, together with the binding characteristics and receptor distribution of [3H]dermorphin in the rat brain. 1 Dermorphin was tested for its relative affinity to μ-, δ-and χ-opioid receptors by determining its potency in displacing the selective μ-receptor ligand [3H]Tyr-DAla-Gly-MePhe-Gly-ol (where Gly-ol=glycinol), the prototypic δ-receptor ligand, [3H]Tyr-DPen-Gly-Phe-DPen (where DPen =β,β-dimethylcysteine) and the χ ligand [3H]ethylketocyclazocine from rat brain and/or guinea pig cerebellum membrane preparations. Inhibitory constant (Ki) values of dermorphin were 0.7 nM. 62 nM and > 5000 nM respectively for μ, δ and χ sites, indicating a selectivity ratio Ki(δ)/Ki(μ) = 88. Under similar conditions, Tyr-DAla-Gly-MePhe-Gly-ol, which is regarded as one of the most selective high-affinity μ-agonist available, exhibited a selectivity ratio of 84. 2 Specific binding properties of tritium-labeled dermorphin (52 Ci/mmol) were characterized in the rat brain. Equilibrium measurements performed over a large range of concentrations revealed a single homogeneous population of high-affinity binding sites (Kd= 0.46 nM; Bmax= 92 fmol/mg membrane protein). 3 Profound differences were observed in the potencies displayed by various selective opiates and opioids ligands in inhibiting the specific binding of [3H]dermorphin. The rank order of potency was in good agreement with that obtained with other μ-selective radiolabeled ligands. 4 Receptor autoradiography in vitro was used to visualize the distribution of [3H]dermorphin binding sites in rat brain. The labeling pattern paralleled that observed using other μ probes. Binding parameters and selectivity profile of [3H]dermorphin on slide-mounted sections were similar to those obtained with membrane homogenates. 5 Finally, intracerebroventricular administration of synthetic dermorphin into mice showed that this peptide is the most potent analgesic known to date, being up to 5 and 670 times more active than β-endorphin and morphine, respectively. Higher doses induced catalepsy. The overall data collected demonstrate that dermorphin is the first among the naturally occurring peptides to be highly potent and nearly specific super-agonist towards the morphine (μ) receptor. The high binding specificity and affinity of dermorphin together with its very low non-specific binding, its high resistance to enzymatic degradation and its ability to cross the blood brain barrier make this natural peptide very attractive for dissecting the role(s) and for identifying molecular and conformational determinants of high-affinity binding to the morphine receptor.

The coupling of high-performance liquid chromatography, gel-permeation, and reversed-phase chromatography with microsequencing proved to be advantageous for the unambiguous determination of specific sites in pro-opiomelanocortin, cleaved by secretory granule lysates of pig anterior pituitary. This system allows the unambiguous identification of a major chymotrypsin-like enzyme activity, optimal at pH 8.0, in the granule preparation, with a specificity directed towards some Phe ↓ X and Tyr ↓ X cleavage sites. The approach used emphasizes the necessity to use methodologies leading to the unambiguous determination of conversion activities.

The postnatal development of cholecystokinin (CCK) binding sites in the rat forebrain and midbrain was studied by in vitro receptor autoradiography. In the majority of structures, the densities of sites were low over the first week after birth, increased until the third week, and decreased over the fourth week to reach adult levels. However, both the rate of increase and the extent of the decrease varied in large proportions among structures. For instance, labeling in the neocortex underwent its largest increase from postnatal day 10, while this increase was already begun at day 7 in the paleocortex. On the other hand, over the fourth postnatal week, the densities could either remain roughly constant (cingulate cortex), slightly decrease (thalamic reticular nucleus), or even return to background levels (pyramidal layer of hippocampus). These different timetables may depend mostly on the differential growth of cells expressing the CCK receptor gene within the developing CNS. The absence of CCK binding sites in most of the regions during the early postnatal period precludes a major role of this peptide in the embryonic development of the rat brain. However, in some regions as the ventromedial hypothalamic nucleus, the endopyriform cortex or the medial nucleus of amygdala, 30–50% of the adult levels were already present at birth. Whether this observation reflects an earlier functional maturation of these structures or a direct participation of the corresponding CCK systems in their development remains to be established.

Taking as a model the N-terminal folding of the cholecystokinin tyrosine-sulfated octapeptide [CCK-8; Asp-Tyr(SO3H)-Met-Gly-Trp-Met-Asp-Phe-NH2] deduced from conformational studies, two cyclic cholecystokinin (CCK) analogues were synthesized by conventional peptide synthesis: Boc-D-Asp-Tyr(SO3H)-Ahx-D-Lys-Trp-Ahx-Asp-Phe-NH2 [compound I (Ahx, 2-aminohexanoic acid)] and Boc-gamma-D-Glu-Tyr(SO3H)-Ahx-D-Lys-Trp-Ahx-Asp-Phe-NH2 (compound II). The binding characteristics of these peptides were investigated on brain cortex membranes and pancreatic acini of guinea pig. Compounds I and II were competitive inhibitors of [3H]Boc[Ahx28,31]CCK-(27-33) binding to central CCK receptors and showed a high degree of selectivity for these binding sites (compound I: Ki for pancreas/Ki for brain, 179; compound II: Ki for pancreas/Ki for brain, 1979). This high selectivity was associated with a high affinity for central CCK receptors (compound I: Ki, 5.1 nM; compound II: Ki, 0.49 nM). Similar affinities and selectivities were found when 125I Bolton-Hunter-labeled CCK-8 was used as a ligand. Moreover, these compounds were only weakly active in the stimulation of amylase release from guinea pig pancreatic acini (EC50 greater than 10,000 nM) and were unable to induce contractions in the guinea pig ileum (to 10(-6) M). The two cyclic CCK analogues, therefore, appear to be synthetic ligands exhibiting both high affinity and high selectivity for central CCK binding sites. These compounds could help clarify the respective role of central and peripheral receptors for various CCK-8-induced pharmacological effects.

Preliminary results on the binding of (/sup 3/H)Boc(Nle/sup 28,31/)CCK/sub 27-33/, designated (/sup 3/H)Boc(diNle)CCK/sub 7/, on mouse brain and rat pancreas membranes are presented. This new ligand for CCK receptors possesses a high specific activity (144 Ci/mmole), and binds in a saturable manner to mouse brain (Kd = 0.49 nM, Bmax = 49 fmoles/mg protein) and rat pancreas (Kd = 4.4 nM, Bmax = 696 fmoles/mg protein). Unlabelled Boc(diNle)CCK/sub 7/ displaces (/sup 125/I)CCK/sub 8/ from its binding sites on mouse brain membranes with a high affinity, slightly superior to that of CCK/sub 8/. The order of potencies to displace (/sup 3/H)Boc(diNle)CCK/sub 7/ from its binding sites was the same in mouse brain and rat pancreas: (/sup 3/H)Boc(diNle)CCK/sub 7/ > CCK/sub 8/, Boc-CCK/sub 7/ > non-sulfated CCK/sub 8/, the pancreas binding sites being more discriminative than the brain binding sites. Thus (/sup 3/H)Boc(diNle)CCK/sub 7/ is a very promising new probe for the characterization of CCK receptors and their interaction with different CCK fragments. 20 references, 4 figures, 1 table.