Antibodies to the N-terminus of human Gastrin-34 react with material in rat and ferret brain

Antibodies specific for the N-terminus of human big gastrin, or NT G34, reveal in immunohistochemistry extensive systems of nerve cell bodies and fibres in the rat and ferret brain. However, when the same antibodies were used in radioimmunoassay of rat and ferret brain tissue extracts they failed to reveal immunoreactive material. At least one of the antibodies used for radioimmunoassay could be shown to react with NT G34 in rat pyloric antral extracts. Antibodies specific for other peptides derived from progastrin failed to reveal the systems demonstrated with the N-terminal G34 antibodies. It is concluded that expression of the gastrin gene is unlikely to account for the present observations. Instead we suggest that a novel peptide with low affinity for NT G34 specific antibodies is found in rat and ferret central neurones.     

Endogenous CNS Expression of Neurotensin and Neurotensin Receptors Is Altered during the Postpartum Period in Outbred Mice

Neurotensin (NT) is a neuropeptide identical in mice and humans that is produced and released in many CNS regions associated with maternal behavior. NT has been linked to aspects of maternal care and previous studies have indirectly suggested that endogenous NT signaling is altered in the postpartum period. In the present study, we directly examine whether NT and its receptors exhibit altered gene expression in maternal relative to virgin outbred mice using real time quantitative PCR (qPCR) across multiple brain regions. We also examine NT protein levels using anti-NT antibodies and immunohistochemistry in specific brain regions. In the medial preoptic area (MPOA), which is critical for maternal behaviors, mRNA of NT and NT receptor 3 (Sort1) were significantly up-regulated in postpartum mice compared to virgins. NT mRNA was also elevated in postpartum females in the bed nucleus of the stria terminalis dorsal. However, in the lateral septum, NT mRNA was down-regulated in postpartum females. In the paraventricular nucleus of the hypothalamus (PVN), Ntsr1 expression was down-regulated in postpartum females. Neurotensin receptor 2 (Ntsr2) expression was not altered in any brain region tested. In terms of protein expression, NT immunohistochemistry results indicated that NT labeling was elevated in the postpartum brain in the MPOA, lateral hypothalamus, and two subregions of PVN. Together, these findings indicate that endogenous changes occur in NT and its receptors across multiple brain regions, and these likely support the emergence of some maternal behaviors.     

Detailed analysis of inflammatory and neuromodulatory cytokine secretion from human NT2 astrocytes using multiplex bead array

Astrocytes are a very important cell type in the brain fulfilling roles in both neuroimmunology and neurotransmission. We have conducted the most comprehensive analysis of secreted cytokines conducted to date (astrocytes of any source) to determine whether astrocytes derived from the human Ntera2 (NT2) cell-line are a good model of human primary astrocytes. We have compared the secretion of cytokines from NT2 astrocytes with those produced in astrocyte enriched human brain cultures and additional cytokines implicated in brain injury or known to be expressed in the human brain. The concentration of cytokines was measured in astrocyte conditioned media using multiplex bead array (MBA), where 18 cytokines were measured simultaneously. Resting NT2 astrocytes produced low levels (～1-30 pg/ml) of MIP1α, IL-6 and GM-CSF and higher levels of MCP-1, IP-10 and IL-8 (1-11 ng/ml) under non-inflammatory conditions. All of these in addition to IL-1β, TNFα, and IL-13, were increased by pro-inflammatory activation (TNFα or IL-1β stimulation). In contrast, IL-2, IL-4, IL-5, IL-7, IL-10, IL-12, LTα, and IFNγ were not detected in astrocyte conditioned media under any of the culture conditions tested. NT2 astrocytes were unresponsive to IL-2 and the adenyl cyclase agonist, forskolin. Interestingly, IFNγ stimulation selectively increased IP-10 secretion only. As astrocytes stimulated with IL-1β or TNFα produced several chemokines in the ng/ml range, we next assessed the chemoattractant properties of these cells. Conditioned media from TNFα-stimulated astrocytes significantly chemoattracted leukocytes from human blood. This study provides the most comprehensive analysis of cytokine production by human astrocytes thus far, and shows that NT2 astrocytes are highly responsive to pro-inflammatory mediators including TNFα and IL-1β, producing cytokines and chemokines capable of attracting leukocytes from human blood. We conclude that in the absence of adult human primary astrocytes that NT2-astrocytes may provide a valuable alternative to study the immunological behaviour of human astrocytes.     

Alterations in regional brain neurotensin concentrations produced by atypical antipsychotic drugs.

Classical antipsychotic drugs, such as haloperidol, have been shown to increase the concentrations of neurotensin (NT) selectively in the nucleus accumbens and caudate nucleus of the rat. Several novel, putative antipsychotic drugs have also been found to produce increases in NT content in one or both of these brain regions. The present study sought to compare the effects of chronic treatment with three clinically efficacious atypical antipsychotic drugs, sulpiride, rimcazole and remoxipride, on regional brain NT concentrations to those of haloperidol. The concentrations of NT in five discrete brain regions were determined by a sensitive and specific radioimmunoassay. As previously reported, haloperidol increased NT concentrations in both the nucleus accumbens and caudate nucleus. Sulpiride and rimcazole produced significant increases in the concentration of NT in the caudate. NT concentrations were unaltered in any brain region by remoxipride at either of the doses tested. These data provide additional evidence for specific increases in regional brain NT concentrations produced by antipsychotic drugs.     

Inactivation of Neurotensin by Rat Brain Synaptic Membranes. Cleavage at the Pro10-Tyr11 Bond by Endopeptidase 24.11 (Enkephalinase) and a Peptidase Different from Proline-Endopeptidase

It was shown previously that the tridecapeptide neurotensin is inactivated by rat brain synaptic membranes and that one of the primary inactivating cleavages occurs at the Pro10-Try11 peptide bond, leading to the formation of NT1-10 and NT11-13. The present study was designed to investigate the possibility that this cleavage was catalyzed by proline endopeptidase and/or endopeptidase 24.11 (enkephalinase). Purified rat brain synaptic membranes were found to contain a N-benzyloxycarbonyl-Gly-Pro-4-methyl-coumarinyl-7-amide-hydrolyzin g activity that was markedly inhibited (93%) by the proline endopeptidase inhibitor N-benzyloxycarbonyl-Pro-Prolinal and partially blocked (25%) by an antiproline endopeptidase antiserum. In contrast, the cleavage of neurotensin at the Pro10-Tyr11 bond by synaptic membranes was not affected by N-benzyloxycarbonyl-Pro-Prolinal and the antiserum. When the conversion of NT1-10 to NT1-8 by angiotensin converting enzyme was blocked by captopril and when the processing of NT11-13 by aminopeptidase(s) was inhibited by bestatin, it was found that thiorphan, a potent endopeptidase 24.11 inhibitor, partially decreased the formation of NT1-10 and NT11-13 by synaptic membranes. In conclusion: (1) proline endopeptidase, although it is present in synaptic membranes, is not involved in the cleavage of neurotensin at the Pro10-Tyr11 bond; (2) endopeptidase 24.11 only partially contributes to this cleavage; (3) there exists in rat brain synaptic membranes a peptidase different from proline endopeptidase and endopeptidase 24.11 that is mainly responsible for inactivating neurotensin by cleaving at the Pro10-Tyr11 bond.     

Immunohistochemical demonstration of neurotensin and tyrosine hydroxylase in iris nerves of the rabbit eye

Summary In previous studies we have provided evidence that intracameral administration of neurotensin (NT), an endogenous tridecapeptide, produces strong miosis in the rabbit. The presence of NT immunoreactivity was investigated in rabbit iris whole mounts by light microscopic immunohistochemistry, and its distribution in the iris compared to that, of tyrosine hydroxylase (TH). A few scattered NT-positive cell bodies were localized in the dilator muscle. Both, the NT cell bodies and processes appeared parallel to the muscle cells. Extensive branching of NT-containing cell processes was observed in connection with the sphincter muscle. These NT-positive fibers formed a dense, randomly oriented network throughout the sphincter muscle cells. The distribution of TH immunoreactivity was similar to that of NT-positive cell processes, except that no TH-positive cell bodies were detected in any of the iris structures examined. Moderate branching of TH-positive fibers was observed in the dilator and sphincter iris muscles. These findings provide neuroanatomical support for an important role of NT in pupillary physiology. Its similar topographical distribution with TH suggests that NT and dopamine may be co-localized, as it has already been described in brain.     

Immunohistochemical demonstration of neurotensin and tyrosine hydroxylase in iris nerves of the rabbit eye

In previous studies we have provided evidence that intracameral administration of neurotensin (NT), an endogenous tridecapeptide, produces strong miosis in the rabbit. The presence of NT immunoreactivity was investigated in rabbit iris whole mounts by light microscopic immunohistochemistry, and its distribution in the iris compared to that, of tyrosine hydroxylase (TH). A few scattered NT-positive cell bodies were localized in the dilator muscle. Both, the NT cell bodies and processes appeared parallel to the muscle cells. Extensive branching of NT-containing cell processes was observed in connection with the sphincter muscle. These NT-positive fibers formed a dense, randomly oriented network throughout the sphincter muscle cells. The distribution of TH immunoreactivity was similar to that of NT-positive cell processes, except that no TH-positive cell bodies were detected in any of the iris structures examined. Moderate branching of TH-positive fibers was observed in the dilator and sphincter iris muscles. These findings provide neuroanatomical support for an important role of NT in pupillary physiology. Its similar topographical distribution with TH suggests that NT and dopamine may be co-localized, as it has already been described in brain.     

NT2 derived neuronal and astrocytic network signalling

A major focus of stem cell research is the generation of neurons that may then be implanted to treat neurodegenerative diseases. However, a picture is emerging where astrocytes are partners to neurons in sustaining and modulating brain function. We therefore investigated the functional properties of NT2 derived astrocytes and neurons using electrophysiological and calcium imaging approaches. NT2 neurons (NT2Ns) expressed sodium dependent action potentials, as well as responses to depolarisation and the neurotransmitter glutamate. NT2Ns exhibited spontaneous and coordinated calcium elevations in clusters and in extended processes, indicating local and long distance signalling. Tetrodotoxin sensitive network activity could also be evoked by electrical stimulation. Similarly, NT2 astrocytes (NT2As) exhibited morphology and functional properties consistent with this glial cell type. NT2As responded to neuronal activity and to exogenously applied neurotransmitters with calcium elevations, and in contrast to neurons, also exhibited spontaneous rhythmic calcium oscillations. NT2As also generated propagating calcium waves that were gap junction and purinergic signalling dependent. Our results show that NT2 derived astrocytes exhibit appropriate functionality and that NT2N networks interact with NT2A networks in co-culture. These findings underline the utility of such cultures to investigate human brain cell type signalling under controlled conditions. Furthermore, since stem cell derived neuron function and survival is of great importance therapeutically, our findings suggest that the presence of complementary astrocytes may be valuable in supporting stem cell derived neuronal networks. Indeed, this also supports the intriguing possibility of selective therapeutic replacement of astrocytes in diseases where these cells are either lost or lose functionality.     

Antinociceptive properties of nasal delivery of neurotoxin-loaded nanoparticles coated with polysorbate-80

Neurotoxin-1 (NT) is an analgesic peptide which is endowed an exceptional specificity of action that blocks transmission of the nerve impulse. The aim of this study was to evaluate the potential application of nanoparticles technology as drug carrier system for the nasal delivery of NT. Mice were administered intranasally (i.n.) with NT (NT-P-NP), free NT solution (F-NT) and intravenously (i.v.) with NT (IV-NT) respectively. The NT levels in animal brain and antinociceptive activity of NT were analyzed. The result on brain transport showed that nanoparticles could exert enhanced delivery of NT into the brain significantly after i.n. administration. The results of antinociceptive activity showed that NT-P-NP increased immobility in the open-field test, both phases of formalin test were significantly inhibited by the NT-P-NP and NT-P-NP significantly inhibited the reaction time to thermal stimuli at 60 and 90 min. Both NT-P-NP and IV-NT were able to inhibit constrictions in acetic acid-induced writhing reaction. These data suggest that NT-loaded nanoparticles coated with polysorbate-80 could generate a significant improvement of drug levels in the brain. Intranasal administration of Neurotoxin-1 entrapped in nanoparticles coated with polysorbate-80 is an attractive alternative to intravenous administration.     

Radioimmunoassay for Lys8, Asn9, neurotensin 8–13: Tissue and subcellular distribution of immunoreactivity in chickens

A sensitive and specific radioimmunoassay (RIA) for Lys⁸, Asn⁹, neurotensin 8–13 (LANT-6) has been developed which utilizes ¹²⁵I-labeled LANT-6 and rabbit antisera raised towards conjugates of synthetic LANT-6 and bovine thyroglobulin. The antiserum described (TG-22) allows the detection of ca 100 fmol of LANT-6 and crossreacts <0.01% with chicken or bovine NT. Dose-response relationships for the native (chicken) and synthetic peptides were indistinguishable. Using this assay the distribution of immunoreactive LANT-6 (iLANT-6) through various tissues of the chicken was studied and compared with that of chicken NT (iNT) determined by RIA. Both iNT and iLANT-6 were found primarily in the brain and gastrointestinal tract, however, their regional distributions were found to differ. Subcellular distribution studies in homogenates of chicken brain indicated that obth iNT and iLANT-6 were associated with synaptosome-like and vesicle-like particles. In homogenates of small intestine, pancreas and colon iNT and iLANT-6 appeared to be within osmotically sensitive, sedimentable particles. Analyses using high pressure liquid chromatography established that chicken iLANT-6 co-eluted with the synthetic peptide and that similar substances were present in extracts of rat brain and intestine. These results are consistent with “messenger” roles for these peptides.     

Differential processing of pro-neurotensin/neuromedin N and relationship to pro-hormone convertases

Neurotensin (NT) is synthesized as part of a larger precursor that also contains neuromedin N (NN), a six amino acid neurotensin-like peptide. NT and NN are located in the C-terminal region of the precursor (pro-NT/NN) where they are flanked and separated by three Lys-Arg sequences. A fourth dibasic sequence is present in the middle of the precursor. Dibasics are the consensus sites recognized and cleaved by endoproteases that belong to the recently identified family of pro-protein convertases (PCs). In tissues that express pro-NT/NN, the three C-terminal Lys-Arg sites are differentially processed, whereas the middle dibasic is poorly cleaved. Pro-NT/NN processing gives rise mainly to NT and NN in the brain, to NT and a large peptide ending with the NN sequence at its C-terminus (large NN) in the gut and to NT, large NN and a large peptide ending with the NT sequence (large NT) in the adrenals. Recent evidence indicates that PC1, PC2 and PC5-A are the pro-hormone convertases responsible for the processing patterns observed in the gut, brain and adrenals, respectively. As NT, NN, large NT and large NN are all endowed with biological activity, the evidence reviewed here supports the idea that post-translational processing of pro-NT/NN in tissues may generate biological diversity.     

Treatment of PC12 cells by nerve growth factor,dexamethasone, and forskolin

Several lines of anatomical, neurochemical, electrophysiological, and behavioral evidence suggest the existence of physiological interactions between neurotensin (NT) and the brain dopaminergic systems. Thus, NT has been shown to exert a neuroleptic-like action and could be implicated in the pathogenesis and treatment of schizophrenia. It is thus of particular importance to develop in vitro cell culture systems as models to study such interactions. Rat adrenal pheochromocytoma PC12 cells, which expressed high levels of tyrosine hydroxylase, were used in the present study. In contrast to rat brain cells in primary cultures, PC12 cells did not express functional NT receptors. However, they were able to express both NTmRNA and NT in response to NGF, forskolin, and dexamethasone. Those neurochemical modifications furthermore may be related to changes in the morphology of the PC12 cells in response to NGF, forskolin, and dexamethasone alone or in combination. These data suggest that PC12 cells may provide a useful model to study in vitro the regulation of both catecholamine and neurotensin phenotypes.     

Neurotrophin-3 and TrkC-immunoreactive neurons in rat dorsal root ganglia correlate by distribution and morphology

Previous studies have shown that a subpopulation of large dorsal root ganglion neurons contains neurotrophin-3 (NT3)-like immunoreactivity. It is not known, however, whether these NT3 immunoreactive neurons also express the high affinity receptor for NT3, trkC. In the present study, the distribution and morphology of trkC immunoreactive neurons have been correlated with those of NT3 immunoreactive neurons in the dorsal root ganglia. Size and segmental distributions of both antigens indicate that they are present in the same group of large sensory neurons. Almost twice the number of these neurons are present in the cervical and lumbar spinal ganglia than in the thoracic. Co-localization study indicates that 94% of NT3 immunoreactive neurons express trkC. Our findings support the proposal that NT3 in these neurons is derived from their peripheral targets rather than synthesized in situ. Special issue dedicated to Dr. Hans Thoenen.     

Immunochemical characterization of neurotensin-like peptides in chicken

Using radioimmunoassay and 3 region specific antisera toward bovine neurotensin (NT), the NT-like peptides in chicken have been shown to differ from NT but to strongly resemble its COOH-terminal region. Three substances were identified, one of which resembled NT biologically and appeared to share 7 or 8 of its COOH-terminal residues. The two other peptides were smaller than NT but seemed to possess 4–6 residue homologies with it. Tissue distribution studies indicated that the chicken pancreas and thymus had unusually high levels of this material (>200 fold that in rat) and that the 3 substances were distributed differently in tissues. Chromatographic studies showed that the peptides obtained from brain, intestine, thymus, and pancreas were similar. These results, demonstrating evolutionary conservation of the COOH-terminal region of NT, are in keeping with the known importance of this region for biological activity. These findings also suggest the existence of an NT-family of peptides serving multiple biological roles.     

Differentiated human NT2-N neurons possess a high intracellular content of myo-inositol

myo-Inositol plays a key role in signal transduction and osmotic regulation events in the CNS. Despite the known high concentrations of inositol in the human CNS, relatively little is known about its distribution within the different cell types. In this report, inositol homeostasis was studied in NT2-N cells, a unique cell culture model of human CNS neurons. Differentiation of precursor NT2 teratocarcinoma cells into NT2-N neurons by means of retinoic acid treatment resulted in an increase in inositol concentration from 24 to 195 nmol/mg of protein. After measurement of intracellular water spaces, inositol concentrations of 1.6 and 17.4 mM were calculated for NT2 and NT2-N cells, respectively. The high concentrations of inositol in NT2-N neurons could be explained by (1) an increased uptake of inositol (3.7 vs. 1.6 nmol/mg of protein/h, for NT2-N and NT2 cells, respectively) and (2) a decreased efflux of inositol (1.7%/h for NT2-N neurons vs. 9.0%/h for NT2 cells). Activity of inositol synthase, which mediates de novo synthesis of inositol, was not detected in either cell type. The observation that CNS neurons maintain a high intracellular concentration of inositol may be relevant to the regulation of both phosphoinositide signaling and osmotic stress events in the CNS.     

NT agonist regulates expression of nuclear high-affinity neurotensin receptors.

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.     

Intermolecular interactions between the neurotensin and the third extracellular loop of human neurotensin 1 receptor

Neurotensin (NT) is a tridecapeptide hormone in the periphery and neurotransmitter in the brain that principally activates three receptor subtypes, named NTS1, NTS2, and NTS3. Since little is known about its structure in the presence of its principal receptor NTS1, we determined it using the key domain of the receptor, i.e. the third extracellular loop. We conclude the following: (i) for the receptor fragment, NT binding modifies its central part, underlying the great flexibility and adaptability of this region; (ii) for bound NT, the extended conformation of its C-terminus is confirmed for the first time in experimental conditions and in the presence of a part of the receptor; and (iii) despite some substitutions, the human receptor residues that are involved in the interaction with NT could be similar to those of the rat receptor which play an important role in NT binding.     

Effect of alpha-lipoic acid supplementation on markers of protein oxidation in post-mitotic tissues of ageing rat

In the present study, we investigated whether DL-alpha-lipoic acid (LA) supplementation could have prooxidant or antioxidant effects on oxidative protein damage parameters such as protein carbonyl (PCO), nitrotyrosine (NT), advanced oxidation protein products (AOPP), and protein thiol (P-SH), as well as oxidative stress parameters such as total thiol (T-SH), non-protein thiol (Np-SH), and lipid hydroperoxide (LHP) in the brain and the skeletal muscle tissue of aged rats. PCO, and NT levels were increased, AOPP and P-SH levels were not changed in the brain tissue of aged rats given LA supplementation. On the other hand, TSH, Np-SH, and LHP levels were decreased in the brain tissue of aged rats given LA supplementation. The levels of the same parameters were not significantly different in the skeletal muscle tissue of aged rats given LA supplementation. The increased levels of protein oxidation markers such as PCO, and NT in the brain tissue of LA-supplemented aged rats compared with non-supplemented aged rats may suggest that oxidative protein damage is increased in LA-supplemented aged rats. We assume that an explanation for our findings regarding LA supplementation on protein oxidation markers in the brain tissue of aged rats may be due to the prooxidant effects of LA. Depending on post-mitotic tissue type and dosage of LA, the prooxidant effects of LA supplementation, should be considered in future studies.