Effects of dexamethasone and forskolin on neurotensin production in rat hypothalamic cultures.

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.

Identification of neurotensin-related peptides in human thymic epithelial cell membranes and relationship with major histocompatibility complex class I molecules.

This study shows the expression at the cell surface of human thymic epithelial cells (TEC) of a neurotensin (NT)-like immunoreactivity. NT radio-immunoassay (RIA) revealed that cultured human TEC contain +/-5 ng immunoreactive (ir) NT/10(6) cells, of which 5% is associated with plasma cell membranes. HPLC analysis of NT-ir present in human TEC showed a major peak of NT-ir corresponding to NT1-13. NT-ir was not detected in the supernatant of human TEC cultures. Using an affinity column prepared with a anti-MHC class I monoclonal antibody, NT-ir-related peptides were retained on the column and eluted together with MHC class I-related proteins. According to the elution time on HPLC of these peptides, they correspond to intact NT1-13, as well as to smaller fragments of NT1-13.

Pharmacological and molecular characterization of the neurotensin receptor expressed in Sf9 cells.

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.

Plasma neurotensin levels in humans: relation to hormone levels in diseases involving the hypothalamo-pituitary-thyroid axis.

This study was aimed to investigate, in humans, the possible relationship between plasma neurotensin (NT) levels and the activity of the hypothalamo-pituitary-thyroid axis. Neurotensin was measured by radioimmunoassay in 14 healthy adult volunteers and in 41 patients among whom 10 were considered as controls and 31 had thyroid dysfunction according to free thyroxine and thyrotropin plasma values. Basal NT levels were not significantly different in healthy adults and in control patients: 9.7 +/- 1.1 fmol/ml (mean +/- SEM) vs 13.3 +/- 2.9 fmol/ml, respectively. In patients with central hypothyroidism the NT level was significantly lower (5.7 +/- 1.2 vs healthy volunteers and controls; p < 0.05) and in patients with peripheral hypothyroidism and hyperthyroidism the NT level was significantly higher (35.9 +/- 12.8 and 29.9 +/- 9.5 fmol/ml, respectively, vs healthy adults (p < 0.01) and vs controls (p < 0.05)). After thyrotropin-releasing hormone (TRH) injection (250 micrograms iv) in nine subjects (two control patients, five patients with hypothyroidism and two patients with hyperthyroidism), NT levels decreased independently of the endocrine status from mean values of 13.4 +/- 8.4 at basal level to 7.3 +/- 0.8 fmol/ml 30 min after injection (p < 0.01 on paired percentage decrease values). These data suggest that plasma NT levels in humans depend upon the pituitary-thyroid status and indicate that TRH could exert a negative regulation on circulating NT levels.

Neurotensin and neuromedin N brain levels after fornix transection: evidence for an efficient neurotensin precursor processing in subicular neurons.

High levels of neurotensin/neuromedin N precursor mRNA, but few if any NT-positive perikarya have been detected in the dorsal subiculum of the adult rat or human hippocampus. This apparent discrepancy was tentatively ascribed to a lack of precursor mRNA translation or to a poor precursor posttranslational processing in neurons of the hippocampus. Another hypothesis is that in long neuronal pathways, maturation of neuropeptide precursors and derived peptides occurs during axonal transport to terminals, a process which accounts for the lack of peptide detection in cell bodies. In order to test this hypothesis, we performed surgical transection of the fornix to interrupt axonal transport of putative NT/NN products arising from the dorsal hippocampus and measured NT and NN levels in different brain regions. In the mamillary bodies, the main projection area of the dorsal subiculum, NN and NT levels were highly reduced 4 or 14 days after the septo-hippocampal transection which was correlated with a slight increase in NN and NT levels in the dorsal hippocampus and the retrosplenial cortex of 4 days lesioned animals. An increase in hypothalamic NN levels was also detected 14 days after the lesion. These data suggest that the peptide precursor processing can take place during the axonal transport, as shown here for neurotensin and neuromedin N from subicular neurons to their efferent brain areas such as the mamillary bodies.

Developmental pattern of neurotensin content in rat hypothalamic neurons cultured in serum-free medium: comparison with in vivo data.

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).

Characterization of neurotensin binding sites on rat mesencephalic cells in primary culture.

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 125I-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 125I-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 125I-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.

Release of vasoactive intestinal peptide from rat brain slices by various depolarizing agents.

The release of vasoactive intestinal peptide (VIP) from rat brain cortical and amygdala slices was studied by using various depolarizing agents such as potassium (K+), veratridine (VER) and batrachotoxin (BTX). The basal release of VIP observed is of the same order of magnitude for both structures and represents less than 0.1% of the tissue content per minute measured by a specific radioimmunoassay. Maximal stimulation obtained with 56 mMK+, 50 microM VER and 1 microM BTX corresponds to a mean 3-fold increase above the basal release of VIP in both cortex and amygdala. When the incubation medium did not contain any calcium, the action of potassium on the release of VIP was suppressed. When tetrodotoxin (1 microM) was added to the incubation medium, the veratridine- and batrachotoxin-induced release of VIP was inhibited whereas K+-induced release was unaffected. These results support the hypothesis that VIP can be a neurotransmitter in the central nervous system.

Plasma neurotensin levels in prepubertal children and adults: possible involvement in the regulation of growth hormone secretion.

The present study investigated a possible relationship between plasma neurotensin (NT) and serum growth hormone (GH) levels after GH-stimulation provocative tests in humans. Samples were obtained from twelve prepubertal children and sixteen normal adult volunteers. Basal plasma NT levels were higher in children with growth delay (19.02 +/- 4.01 fmol/ml) (mean +/- SEM) than in normal adults (6.13 +/- 1.1 fmol/ml) (p < 0.001). Basal GH levels in children (1.52 +/- 0.06 ng/ml) were not different from those in adults (0.60 +/- 0.41 ng/ml). After stimulation of GH secretion, NT values decreased when GH peaked, and increased when GH levels diminished. These data suggest that plasma NT levels may be involved in the regulation of GH secretion, as a peripheral signal, probably through modulation of somatostatin release from the median eminence.

[Radioautographic localization of neuropeptide receptors in the central nervous system]. / Localisation par radioautographie des récepteurs des neuropeptides dans le système nerveux central.

The first step of any physiological effect of a neuropeptide (NP) is its recognition by specific receptor sites. The very organization of the central nervous system (CNS) does not permit a precise localization of these binding sites by conventional binding assays. The aim of the present paper is to describe in detail a recently developed in vitro methodology for the localization, visualization and quantitation of specific binding sites for various NP such as TRH, neurotensin and vasoactive intestinal peptide (VIP) in the rat CNS. The combination of this autoradiographic technique with radioimmunological measurements of NP, reveals that the endogenous distribution of THR, for example, in various brain regions, is not correlated with the presence of its binding sites. In vitro autoradiography may also be used to study the neurotransmitter/neuromodulatory role of NP in the CNS. This point will be illustrated by the effect of VIP on serotonin binding sites in both rat suprachiasmatic nucleus and hippocampal formation. Besides, the importance of the endocrine environment of the target tissue for NP action will also be discussed.

[Radioautographic and quantitative study of insulin binding sites in the rat brain]. / Etude radioautographique et quantitative des sites de liaison de l'insuline dans le cerveau de rat.

In the present study, we describe the specificity and the autoradiographic distribution of insulin binding sites in the rat central nervous system (CNS) after in vitro incubation of brain sections with [125I]-14A insulin. Increasing concentrations of unlabeled insulin produced a dose-dependent inhibition of [125I]-insulin binding which represented 92 +/- 2% displacement with 3 X 10(-5) M, whatever the brain sections tested. Half-maximum inhibition with native insulin was obtained with 2.2 X 10(-9) M, with 10(-7) M proinsulin whereas glucagon had no effect. Under our experimental conditions, no degradation of [125I]-insulin was observed. Autoradiograms obtained by apposition of LKB 3H-Ultrofilm showed a widespread distribution of [125I]-insulin in rat CNS. However, quantitative analysis of the autoradiograms with 10(-10) M of labeled insulin, showed a high number of [125I]-insulin binding sites in the choroid plexus, olfactory areas, in both cerebral and cerebellar cortices, the amygdaloid complex and in the septum. In the hippocampal formation, the dorsal dentate gyrus and various subfields of CA1, CA2 and CA3 were labeled. Moreover, arcuate, dorso- and ventromedial nuclei of the hypothalamus contained high concentrations of [125I]-insulin whereas a low density was observed in the mesencephalon. The metabolic role of insulin in the CNS is supported by the large distribution of insulin binding sites in the rat brain. However, the presence of high affinity binding sites in selective areas involved in perception and integrative processes as well as in the regulation of both feeding behavior and neuroendocrine functions, suggests a neuromodulatory role of insulin in the brain.

Apparent functionality but impractical quantification of neurotensin receptors on human peripheral lymphocytes.

Neurotensin (NT), a neuromodulator, is also thought to play an immunomodulatory role. We sought to confirm the effects of NT on proliferation in human peripheral blood lymphocytes (PBLs; n = 10), to characterize the binding properties of the NT receptor using Scatchard analysis, and to measure NT receptors using blood volumes (50 ml) applicable to clinical investigation. Incubation of phytohemagglutinin-stimulated PBLs with NT (10[-10] to 10[-13] M) in the presence of 0.07% normal human serum (72 h at 37 degrees C) significantly enhanced proliferation (p < 0.001), which is consistent with PBLs possessing functional NT receptors. However, the various experimental conditions tested constantly yielded low specific NT binding to human PBLs, and hence Scatchard analysis was impossible for 50-ml blood samples. Our data confirm the existence of a link between NT and the immune system and support a physiological significance for this link. However, measurement of NT receptor binding in readily available cells proved unsuitable for clinical investigation.

Effects of growth hormone administration on neurotensin release in children with growth delay.

Neurotensin (NT), a tridecapeptide originally isolated from bovine hypothalamus, has numerous actions on endocrine functions. Since intravenous injection of NT in the rat stimulated the release of growth hormone (GH) among several pituitary hormones, the aim of our study was to investigate in humans the effects of GH injection on NT plasma levels. Plasma samples were obtained from 13 children with growth delay (7 boys and 6 girls; age range 5 years 1 month-14 years 1 month; mean +/- SE 10 years 9 months +/- 7 months) to evaluate NT and GH values before treatment and 4, 12 and 24 h after a subcutaneous rhGH injection (0.15 IU/kg). Plasma was extracted on a SEP-PAC C18 column and NT was eluted with propanol. NT concentrations were measured by a specific RIA and expressed as fmol/ml plasma. GH (ng/ml) and somatomedin C (SMC; U/ml) were evaluated by RIA using commercial kits. Free fatty acids (FFA; mEq/l) were measured using a colorimetric peroxidase technique. Before GH administration, NT levels were 7.19 +/- 1.01 fmol/ml. A significant increase in NT values was found 4 h (36.5 +/- 9.62, p < 0.001), 12 h (40.85 +/- 6.64, p < 0.001) and 24 h (19.5 +/- 3.48, p < 0.05) after GH injection. This increase was significantly correlated with the circulating GH levels 4 h after GH administration and with the circulating SMC levels 24 h after GH administration. No correlation was found between NT and FFA values.(ABSTRACT TRUNCATED AT 250 WORDS)

Mesencephalic dopaminergic neurons in primary cultures express functional neurotensin receptors.

The cellular distribution and functional aspects of neurotensin (NT) binding sites in rat mesencephalic cells in primary culture were investigated by an original approach combining anatomical and biochemical studies. Using a double-labeling protocol combining 125I-NT receptor radioautography and tyrosine hydroxylase (TH) immunocytochemistry, we obtained the first direct visualization of NT binding sites on TH-immunoreactive neurons. Eighty percent of the TH neurons were endowed with NT binding sites, which can be observed on both cell bodies and processes. TH-immunoreactive neurons were characterized as dopaminergic neurons by their ability to take up dopamine in a benztropine- and nomifensine-sensitive manner. In the mesencephalic cultures, NT increased potassium-evoked release of tritiated dopamine, and the relative potencies of various NT-related peptides to increase dopamine release were in good agreement with their abilities to bind to NT sites. These results show for the first time that cultured rat mesencephalic dopaminergic cells express functional NT receptors. Finally, the specificity and distribution of NT receptors on dopaminergic neurons in primary culture are quite similar to what was observed in the adult rat brain using pharmacological and radioautographic approaches. These data indicate that NT can influence the activity of dopaminergic neurons at very early stages of the rat brain development.

[Effect of interleukins and somatomedins on the production of neurotensin by cell line SH-SY5Y derived from human neuroblastoma]. / Effet des interleukines et des somatomédines sur la production de neurotensine par la lignée cellulaire SH-SY5Y dérivée d'un neuroblastome humain.

The goal of this study was to investigate the regulation by insulin-like growth factors 1 and 2, and interleukins on the production of neurotensin in the SH-SY5Y cell line derived from a human neuroblastoma. Cultures were performed in RPMI1640 culture medium with heated foetal calf serum 12%. After 24 hrs. of fasting without serum, interleukins-1 alpha, IL-2, IL-4 and insulin-like growth factors 1 and 2 were added. Results showed: 1) A mitogenic effect of ILs (p < 0.001) and of IGFs (p < 0.001). 2) The presence of neurotensin in HCl0.1N cellular extracts (0.06 fmol/micrograms protein). 3) The increase of cellular neurotensin content in the presence of IL-4 (560%), IL-2 (480%), IGF-1 (610%) and IGF-2 (200%). Our results indicate that the human neuroblastoma cell line SH-SY5Y produces neurotensin and that ILs and IGFs act in vitro to modulate this production.

Release of vasoactive intestinal peptide from rat jejuno-ileum in vitro. Effect of various depolarizing agents.

Vasoactive intestinal peptide (VIP) can be released in vitro from intestinal slices under veratridine and batrachotoxin depolarization, whereas potassium depolarization has no effect. The lack of an effect of potassium observed in this peripheral preparation is different from the positive action described for it in the CNS. The present data suggest that VIP can be released through different mechanisms in the peripheral and central nervous system.

Effects of ion channel blockers and phorbol ester treatments on [3H]dopamine release and neurotensin facilitation of [3H]dopamine release from rat mesencephalic cells in primary culture.

In this work, we tested the effect of ion channel blockers and of phorbol ester treatments on [3H]dopamine ([3H]DA) release and neurotensin (NT)-induced facilitation of [3H]DA release from cultures of rat fetal mesencephalic cells. The potassium channel blockers tetraethylammonium and 4-aminopyridine increased basal [3H]DA release and decreased K(+)-evoked [3H]DA release, whereas apamin was without effect. K(+)-evoked [3H]DA release was decreased by omega-conotoxin and nifedipine, totally suppressed by cadmium, and unaffected by amiloride. These results show the differential sensitivity of [3H]DA release to blockade of various ion channels and suggest the involvement of N-type, L-type, and non-L-non-N-type, but not T-type, voltage-sensitive calcium channels in K(+)-evoked release. Phorbol 12-myristate 13-acetate increased both spontaneous and K(+)-evoked [3H]DA release, suggesting a modulatory action of protein kinase C on DA release in this system. Unexpectedly, however, the effects of the phorbol ester were not counteracted by the protein kinase C inhibitors H7, staurosporine, or polymyxin B. NT-induced facilitation of K(+)-evoked [3H]DA release was insensitive to most of the ion channel blockers, except cadmium (64% decrease in NT effect), suggesting that the corresponding potassium and calcium channels were not involved in the effect of NT on [3H]DA release in this system. The NT effect was totally suppressed by phorbol ester treatments, indicating a possible desensitization of the corresponding transduction mechanisms after protein kinase C activation.

Increased plasma neurotensin concentrations in patients with Parkinson's disease.

Plasma neurotensin (NT) was measured by radioimmunoassay in propanol extracted and unextracted plasma from 16 parkinsonian patients (four before treatment) and 16 age and sex matched controls. Mean plasma NT concentrations were consistently higher in parkinsonian patients than in controls and higher in the four untreated patients than in levodopa treated patients suggesting that plasma NT measurement may represent an easy detectable additional index in diagnosing parkinsonism and provides a novel approach to research in this field.