Prevention of testosterone aromatization in the female rat pups by perinatal treatment with 1,4,6 androstatriene-3,17-dione (ATD) induces an important defeminization as shown by a reduction of fluctuations of LH release after castration and estradiol implantation. The fact that, under our in vitro experimental conditions, ATD is able to displace testosterone binding in the hypothalamus whereas estradiol does not, confirms the hypothesis that ATD acts on aromatase. The most attractive explanation for the defeminization effect of ATD is then an estrogen-like action of ATD.

In this work, we tested the effect of ion channel blockers and of phorbol ester treatments on [ 3 H]dopamine ([ 3 H]DA) release and neurotensin (NT)‐induced facilitation of [ 3 H]DA release from cultures of rat fetal mesencephalic cells. The potassium channel blockers tetraethylammonium and 4‐aminopyridine increased basal [ 3 H]DA release and decreased K + ‐evoked [ 3 H]DA release, whereas apamin was without effect. K + ‐evoked [ 3 H]DA release was decreased by ω‐conotoxin and nifedipine, totally suppressed by cadmium, and unaffected by amiloride. These results show the differential sensitivity of [ 3 H]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 [ 3 H]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 [ 3 H]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 [ 3 H]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.

The presence of endothelin binding sites in the human placenta raises the question of the precise localization of these receptors on well defined placental constituents. In order to find an answer to this problem various approaches were used. Specific binding sites for [125I] endothelin-1 (ET-1) were identified on human term placenta, not only on membranes of smooth muscles stem villi vessels, but also on trophoblastic plasma membranes prepared from trophoblast in culture. Scatchard analysis of binding data revealed a single class of high affinity binding sites with Kd values of 26 +/- 4 pmol/L for stem villi vessels and 126 +/- 4 pmol/L for trophoblast in culture, with maximum binding capacities of 681 +/- 61 and 224 +/- 53 fmol/mg protein, respectively. The anatomical localization of these binding sites was determined by in vitro autoradiography. Autoradiograms obtained from placental sections incubated with [125I]ET-1 indicate that [125I]ET-1 high affinity binding sites exist on placental stem villi vessels and on the trophoblastic layer of the villi. The latter localization was also found on autoradiograms of trophoblast in culture. The human placental syncytiotrophoblast is a polarized epithelium with the microvillous membrane, facing maternal blood space and the basal plasma membrane, facing fetal circulation. [125I]ET-1 high affinity binding sites are present on both membranes but the number of binding sites is higher on the basal plasma membrane. These findings lead to the suggestion that ET-1 may be involved in the regulation of the feto-placental circulation and may subserve specific trophoblastic functions.

Annals of the New York Academy of SciencesVolume 668, Issue 1 p. 217-231 Interaction between Neurotensin and Dopamine in the Brain Morphofunctional and Clinical Evidencea WILLIAM ROSTÈNE, Corresponding Author WILLIAM ROSTÈNE INSERM U.339, Hǒpital Saint-Antoine, 75571 Paris Cedex 12, FranceAddress for correspondence: INSERM U.339, Hǒpital Saint-antoine, 184, rue du Fg. St. Antoine, 75571 Paris Cedex 12, France.Search for more papers by this authorALINE BROUARD, ALINE BROUARD INSERM U.339, Hǒpital Saint-Antoine, 75571 Paris Cedex 12, FranceSearch for more papers by this authorCORINNE DANA, CORINNE DANA INSERM U.339, Hǒpital Saint-Antoine, 75571 Paris Cedex 12, FranceSearch for more papers by this authorYOSHINORI MASUO, YOSHINORI MASUO INSERM U.339, Hǒpital Saint-Antoine, 75571 Paris Cedex 12, FranceSearch for more papers by this authorFRANCE AGID, FRANCE AGID INSERM U.289, Hǒpital de la Salpétrière, 75651 Paris Cedex 13, FranceSearch for more papers by this authorMICHELINE VIAL, MICHELINE VIAL INSERM U.339, Hǒpital Saint-Antoine, 75571 Paris Cedex 12, FranceSearch for more papers by this authorANNE-MARIE LHIAUBET, ANNE-MARIE LHIAUBET INSERM U.339, Hǒpital Saint-Antoine, 75571 Paris Cedex 12, FranceSearch for more papers by this authorDIDIER PELAPRAT, DIDIER PELAPRAT INSERM U.339, Hǒpital Saint-Antoine, 75571 Paris Cedex 12, FranceSearch for more papers by this author WILLIAM ROSTÈNE, Corresponding Author WILLIAM ROSTÈNE INSERM U.339, Hǒpital Saint-Antoine, 75571 Paris Cedex 12, FranceAddress for correspondence: INSERM U.339, Hǒpital Saint-antoine, 184, rue du Fg. St. Antoine, 75571 Paris Cedex 12, France.Search for more papers by this authorALINE BROUARD, ALINE BROUARD INSERM U.339, Hǒpital Saint-Antoine, 75571 Paris Cedex 12, FranceSearch for more papers by this authorCORINNE DANA, CORINNE DANA INSERM U.339, Hǒpital Saint-Antoine, 75571 Paris Cedex 12, FranceSearch for more papers by this authorYOSHINORI MASUO, YOSHINORI MASUO INSERM U.339, Hǒpital Saint-Antoine, 75571 Paris Cedex 12, FranceSearch for more papers by this authorFRANCE AGID, FRANCE AGID INSERM U.289, Hǒpital de la Salpétrière, 75651 Paris Cedex 13, FranceSearch for more papers by this authorMICHELINE VIAL, MICHELINE VIAL INSERM U.339, Hǒpital Saint-Antoine, 75571 Paris Cedex 12, FranceSearch for more papers by this authorANNE-MARIE LHIAUBET, ANNE-MARIE LHIAUBET INSERM U.339, Hǒpital Saint-Antoine, 75571 Paris Cedex 12, FranceSearch for more papers by this authorDIDIER PELAPRAT, DIDIER PELAPRAT INSERM U.339, Hǒpital Saint-Antoine, 75571 Paris Cedex 12, FranceSearch for more papers by this author First published: October 1992 https://doi.org/10.1111/j.1749-6632.1992.tb27352.xCitations: 44 a This work was supported by INSERM, the Ministère de la Recherche et de la Technologie (MRT fellowship to A.B.), ARC (fellowship to C.D.), and by the Canon Foundation (fellowship to Y.M.). AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Citing Literature Volume668, Issue1The Neurobiology of NeurotensinOctober 1992Pages 217-231 RelatedInformation

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.

Neurotensin (NT) is a biologically active peptide originally isolated from bovine hypothalamus. Since the presence of NT has been demonstrated in human plasma, its possible source from blood cells cannot be excluded. NT was measured by radioimmunoassay in conditioned medium from incubated peripheral blood lymphocytes (PBL) and in 0.1 M HCl cellular extracts from normal adult subjects (n = 9). Activated cell cultures were tested at different cellular concentrations in RPMI-1640 medium containing 1 % normal human serum and PHA-M (4.8 g/1) for various incubation times. NT levels in the conditioned medium increased with the incubation time (at 5 x 10(6) cells, NT increased from 37 fmol after 18 h to 200 fmol after 96 h of incubation). Moreover, NT levels were significantly correlated (r = 0.940, p < 0.02) with the number of cells from 0.65 at 12 x 10(6) cells. In the acid cellular extracts of the 5 subjects studied, NT levels corresponded to a mean value of 727 fmol/mg protein. Indeed, NT contents of PBL were 4 times higher in actived PBL than in nonactivated cells. Our data suggest possible production of NT by PBL.

1. In the present study we examined the in vitro effect of vasoactive intestinal peptide (VIP) on spontaneous contractions in both inner and outer layers of non-pregnant human myometrium. A dose-dependent relaxation was observed, but with a marked difference in sensitivity to VIP between the two layers, with an IC50 value of 1 x 10(-8) and 1 x 10(-5) mol L in the outer and inner layers, respectively. 2. We also established that VIP did not directly stimulate the adenylate cyclase activity. The only slight stimulations were observed in non-initial rate conditions. The maximal response of this indirect effect was obtained for VIP concentrations between 1 x 10(-9) and 1 x 10(-8) mol/L and this occurred to the same extent (an approximately 1.4-fold increase) in both layers. However this response is specific, since structurally related peptides such as glucagon, gastric inhibitory polypeptide (GIP), secretin, or human growth hormone-releasing factor (hGRF) had no effect in our preparations. 3. Autoradiographic studies revealed that specific VIP binding sites were located on the vascularization of the intermediate vascular layer and on arterioles and venules distributed in the inner and outer myometrial layers. They were also present in the endometrium, but not on smooth muscle cells of either layer. 4. Such observations could provide evidence for another signal transduction pathway to mediate the biological effect of VIP. An additional intermediate step on the vascularization distributed in all of the muscle cannot be excluded.

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.

The presence of Vasoactive Intestinal Peptide (VIP) has been reported in human placenta, a non-innervated organ. However, the exact origin of this neuropeptide, the precise localization of its binding sites, and the mechanism(s) of action remain tobe clarified. The 125I-VIP binding to placental slices was saturable, and unlabeled VIP was able to compete in a dose-dependent manner with an IC50 value of 5.2±1.3×10−10M.

The distribution of specifically labeled neurotensin (NT) binding sites was examined by light and electron microscopic radioautography in the ventral tegmental area (VTA) and nucleus interfascicularis of the rat following incubation of lightly prefixed midbrain slices with the monoiodinated ligand, 125I-(Tyr3)-NT. Film radioautograms of whole 125I-NT-incubated slices exhibited intense NT displaceable binding throughout the VTA and interfascicular nucleus. In light microscopic radioautographs from 1-microns-thick sections taken from the surface of the slices, the label was found to be present both inside and outside neuronal perikarya. Probability circle analysis of silver grain distribution in electron microscopic radioautographs confirmed that a significant proportion (greater than 20%) of the specifically labeled binding sites was intraneuronal. The frequent association of these sites with profiles of rough endoplasmic reticulum or Golgi apparatus suggested that they corresponded in part to receptors undergoing synthesis and/or glycosylation. The remainder was associated with neuronal and/or glial plasma membranes, as attested by comparing the distribution of grains overlying apposed cellular elements with the distribution of hypothetical grains originating from randomly distributed membrane bound radioactive sources. Although the resolution of the technique did not make it possible to ascribe labeled membrane-bound receptors to either one of the apposed plasma membranes, their frequent association with interfaces involving the plasmalemma of perikarya and dendrites, together with the occurrence of silver grain alignments along the membrane of certain somata and dendrites suggested that a proportion of them was associated with the perikarya and dendrites of a subpopulation of ventral tegmental neurons. Interestingly, these perikaryal and dendritic receptors were not exclusively present on, or even concentrated opposite, abutting axon terminals but instead were more or less evenly distributed along the plasma membrane. Only an exceedingly small proportion was found to be associated with synaptic junctions. Such a low incidence makes it unlikely that only the synapse-linked binding sites correspond to functional receptors. On the contrary, the dispersion of labeled receptors seen here along the plasma membrane of presumptive dopamine neurons suggests that NT acts mainly in a paracrine or parasynaptic fashion in the ventral midbrain tegmentum.

The binding of [3H]dihydrotetrabenazine, a specific ligand of the monoamine transporter present on serotonin and catecholamine synaptic vesicles, was studied on rat brain sections. The characteristics of binding (Kd = 5.0nM, k1 = 0.13 × 106M−1 s−1;k−1 = 0.66 × 10−3s−1) were similar to those previously observed on tissue homogenates. The rostrocaudal topographical distribution of dihydrotetrabenazine binding sites was analysed by quantitative autoradiography. High labelling was observed in regions richly innervated by monoaminergic systems: dopamine in the striatum and olfactory tubercles, noradrenaline in the striatal fissure and in the paraventricular and dorsomedial hypothalamus and serotonin in the lateral septum, islands of Calleja and suprachiasmatic nucleus. Cell bodies were also labelled in the substantia nigra and ventral tegmental area (dopamine), in locus coeruleus (noradrenaline) and in raphe nucleus (serotonin). The pituitary gland (particularly the neural lobe) and the pineal gland were also labelled. Low labelling was observed in various areas of the cerebral cortex and in the cerebellum. Unilateral 6-hydroxydopamine lesion of the substantia nigra dramatically reduced [3H]dihydrotetrabenazine labelling in the ipsilateral striatum. Moreover, ketanserin has recently been shown to possess a nanomolar affinity for the vesicular monoamine transporter and autoradiographic localization of brain monoaminergic synaptic vesicles was also obtained by means of the derivative 7-amino-8-[125I]iodoketanserin in the presence of 5-hydroxytryptamine2 and α1 antagonists, although the non-specific labelling was higher than with [3H]dihydrotetrabenazine.

This chapter discusses the imaging of the neuropeptide–neurotransmitter interactions. The chapter focuses on the data obtained by applying quantitative receptor autoradiography (ARG) and computer image analysis to the study of interactions among neuropeptides and classical neurotransmitters—neurotensin, dopamine (DA), vasoactive intestinal peptide, and serotonin. Communication among nerve cells involves more than the direct excitation and inhibition of neuronal firing by neurotransmitters. Several other chemical messengers along with blood-borne hormones modulate the response properties and hence the functional capacity of nerve cells. Modulation may take place directly at the primary level, that is, receptor site, and then involves changes in activation of intracellular second messengers regulating the phosphorylation of specific proteins by protein kinases. Modulation can also occur through regulation of genomic activity and expression of gene products involved in nerve cell function. The development of new techniques of brain imaging has enabled to understand the multifaceted brain interactions and how they may underlie behavioral responses and/or neurological disorders.

The topographie distribution of specifically labeled neurotensin binding sites was examined by light microscopic radioautography in rat brain sections incubated with monoiodo [125I]Tyr3-neurotensin. Preliminary experiments indicated that under the present experimental conditions [125I]neurotensin specifically binds to a single apparent population of sites with a dissociation constant of 7.7 ± 0.3nM, and that fixation of the labeled sections with glutaraldehyde ensures regionally proportional retention of more than 70% of bound [125I]neurotensin molecules. High concentrations of [125I]neurotensin binding sites were detected in the olfactory bulb and tubercle, parts of the neocortex, the lateral septum, the diagonal band of Broca, the caudate putamen, the amygdala, the dentate gyrus, the anterior dorsal nucleus of the thalamus, the suprachiasmatic nucleus of the hypothalamus, the medial habenula, the zona incerta, the substantia nigra and the ventral tegmental area. In certain areas, such as in the diagonal band of Broca, the substantia innominata, the nucleus basalis and the pars compacta of the substantia nigra, discrete accumulations of silver grains were apparent over neuronal perikarya and their proximal dendrites. In most areas, however, the label appeared more or less uniformly distributed over nerve cell bodies and surrounding neuropil. In several instances, the labeling conformed with the distribution of cell bodies of origin and terminal arborizations of specific projection systems, suggesting that neurotensin receptors might be distributed both proximally and distally on the plasma membrane of certain neurons.

Rat brain sections, located at the hippocampal level, were used to study the effect of bilateral adrenalectomy, with or without corticosterone treatment, on the number and affinity of corticosteroid binding sites. Adrenalectomy induces an increase of corticosterone receptor binding sites whereas adrenalectomy followed by in vivo corticosterone treatment produces a 50% decrease of binding site number. Increases and decreases of binding site number were not associated with a significant modification of the affinity for corticosterone. The present data show that in vivo corticosterone modulates its own number of binding sites demonstrated by in vitro binding on brain sections, in a manner which is reminiscent of changes in cytosol receptors demonstrated by conventional biochemical methods. Thus, this in vitro method provides an alternative way to study the plasticity of hippocampal glucocorticoid receptors.

The characteristics and topographical distribution of monoiodo 125I-Tyr3-neurotensin (NT) binding sites in normal human brain tissue were studied on brain sections and by quantitative autoradiography. Sections at the level of the substantia nigra show a dissociation constant and maximal binding capacity of 4.8 +/- 0.8 nM and 70 +/- 7 fmol/mg protein, respectively. High density of 125I-NT binding sites were mainly found in dopaminergic (DA)-rich areas such as the substantia nigra, the ventral tegmental area, the striatum and the nucleus accumbens, further supporting an interaction between NT and DA neurons in human brain.

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.