Quantitative autoradiographic localization of neurotensin binding sites in lean and obese Zucker rats.
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Animals↗
Biomedical subjects
M Dussaillant
Publications and source records attributed to M Dussaillant.
Adrenal steroid modulation of vasoactive intestinal peptide effect on serotonin1 binding sites in the rat brain shown by in vitro quantitative autoradiography.
In the present work we demonstrate by means of quantitative in vitro autoradiography that the vasoactive intestinal peptide (VIP) is able to increase the number of serotonin1 (5-HT1) binding sites in the dorsal subiculum of the rat hippocampus and to decrease them in the suprachiasmatic nucleus (SCN). Bilateral adrenalectomy (ADX) for 6 days counteracted the stimulatory effect of VIP on 5-HT1 binding sites in the dorsal subiculum, but did not modify the inhibitory effect of the peptide in the SCN. Moreover, ADX increased 5-HT1 binding sites in response to VIP in various subfields of the hippocampus as well as in the superior colliculus and in the dorsal lateral septum, but this effect was not observed in normal or in ADX rats bearing a corticosterone implant. The present data are suggestive of a possible interaction between VIP and 5-HT in the regulation of the SCN and of a modulatory role of adrenal steroids in VIP activity in the hippocampal formation.
Adrenalectomy↗
Chemical specificity of the effects of branched chain amino acids on glucagon and insulin release in the suckling rat.
The work presented here aims at investigating the in vitro relationship between amino acids and pancreatic hormonal release in the suckling rat. Among the 20 most usual amino acids, 12 of them were tested on perifused pieces of pancreas in 5.5-day-old rats at a low glucose concentration. Their effects were compared to those induced by a 20 amino acid mixture. A 20 amino acid mixture (10.3 mM) stimulated glucagon and insulin release (154 and 208%, respectively). In the mixture, the 4 pooled amino acids Ala-Gly-Ser-Thr (at 1.4 instead of 4.2 mM) did not induce further modification of the glucagon or insulin release. However, the 3 branched chain amino acids Ile-Leu-Val (at 2.9 instead of 1.4 mM) or the following amino acids Arg-Asn-Phe-Pro-Tyr (at 3.2 instead of 1.6 mM) modulated glucagon or insulin release in the presence of the amino acid mixture. In the absence of Ile-Leu-Val, the amino acid mixture had no effect on the insulin release: 122 compared to 208%. This seems specific to the chemical nature of Ile-Leu-Val since in the absence of Arg-Asn-Phe-Pro-Tyr, the amino acid mixture was still effective on the insulin release (178 compared to 208%). In the absence of Ile-Leu-Val, the amino acid mixture was effective on glucagon release, and revealed the cellular specificity of the branched chain amino acid action.
Amino Acids↗
[Radioautographic localization of neuropeptide receptors in the central nervous system].
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.
Animals↗
In vitro binding of tritiated glucocorticoids directly on unfixed rat brain sections.
We describe a new technique for measuring specific in vitro binding of tritiated adrenal steroids on unfixed cryostat brain sections. The specific binding of [3H]corticosterone represents about 70% of the initial binding. Kinetic studies show that specific binding for [3H]corticosterone reaches equilibrium after 15 min incubation at room temperature. Scatchard analysis of [3H]corticosterone in vitro binding gives a linear plot with an apparent dissociation constant (Kd) and a number of binding sites (Bmax) in the range of 10(-8) M and 100 fmol/mg protein, respectively. [3H]Dexamethasone binding under the same conditions gives a similar Kd and a Bmax of 55 fmol/mg protein. The order of potency for the relative binding affinity for [3H]corticosterone labeled sites is as follows: corticosterone greater than progesterone, dexamethasone, RU 38486 (a "pure" antiglucocorticoid), RU 26988 (a "pure" glucocorticoid), aldosterone greater than estradiol, testosterone. Anatomical studies reveal that sections at the level of the hippocampus bind more [3H]corticosterone and [3H]dexamethasone in vitro than more rostral sections taken at the level of the septum. Adrenalectomy increases the capacity of [3H]corticosterone to bind to these sites and perfusion of the brain to remove transcortin and other blood proteins does not modify [3H]corticosterone binding. We conclude that it is possible to measure in unfixed frozen brain sections glucocorticoid binding sites.
Adrenalectomy↗
In vitro biochemical characterization and autoradiographic distribution of 3H-thyrotropin-releasing hormone binding sites in rat brain sections.
In the present study, we describe the biochemical characteristics and the autoradiographic distribution of thyrotropin-releasing hormone (TRH) receptors in the rat central nervous system (CNS) after in vitro incubation of brain slices with 3H-TRH. Scatchard analysis showed that, in the range of concentrations tested (0.7-35 nM), 3H-TRH bound to a single-class of receptors with a dissociation constant of 6 nM and a number of binding sites of 20 fmol/mg protein. Increasing concentrations of unlabeled TRH produced a dose-dependent inhibition of 3H-TRH binding. The only analogue as potent as TRH to displace 3H-TRH binding was 3-Me-TRH, whereas 1-Me-TRH or TRH-free acid as well as pGlu-His, pGlu-Pro-NH2 or His-Pro-diketopiperazine were ineffective. Neither Luteinizing hormone-releasing hormone (LHRH), neurotensin, somatostatin, D-Ala-Met-enkephalin nor VIP showed any significant affinity for TRH binding sites. Autoradiograms obtained by apposition of LKB 3H-Ultrofilm showed that the highest concentrations of 3H-TRH binding sites were found in the ventral dentate gyrus of the hippocampal formation, the lateral amygdaloid nucleus, the nucleus accumbens, and the thalamic paraventricular nucleus. The biochemical characterization of 3H-TRH binding in brain sections is in good agreement with previous reports on membrane preparations and the autoradiographic localization of the binding sites provides anatomical support for the effects of TRH in the CNS.
Amygdala↗
[Binding of tritiated corticosterone by brain sections of adrenalectomized rats].
In the present work we describe a new technique which permits us to study the specific binding of tritiated corticosterone in brain sections of adrenalectomized Rats. Under these conditions, the specific binding of the glucocorticoid represents 60 to 70% of the initial binding. The apparent dissociation constant and the number of binding sites, determined by Scatchard analysis, are in the range of 10(-8) M and 100 fmoles/mg of protein respectively.
Adrenalectomy↗
Rapid inhibition by somatostatin of vasoactive intestinal peptide-induced prolactin secretion by rat pituitary cells. Relationship to cyclic AMP accumulation.
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1-Methyl-3-isobutylxanthine↗
Rapid glucocorticoid inhibition of vasoactive intestinal peptide-induced cyclic AMP accumulation and prolactin release in rat pituitary cells in culture.
Vasoactive intestinal peptide (VIP) stimulates both adenosine 3',5'-cyclic monophosphate (cAMP) accumulation and prolactin release in normal rat pituitary cells in culture. cAMP accumulation is significant (P less than 0.01) at VIP concentrations as low as 1 nM and reaches a maximum with 0.1 microM. Addition of dexamethasone as early as 15 min before VIP inhibits VIP stimulation of both cAMP production and PRL secretion. The rapid inhibition is dose-dependent: it appears at doses as low as 0.01 pM and is complete at 1 pM dexamethasone. Increasing concentrations of dexamethasone induce a noncompetitive type of inhibition, as shown by the decrease in Vmax with no change in the apparent Km for VIP. Cycloheximide (1 mM) counteracts the inhibitory effect of dexamethasone on VIP-induced cAMP production, which suggests the involvement of a rapid protein synthesis mechanism. Ru-26988, a specific glucocorticoid devoid of any mineralocorticoid activity and which does not bind to intracellular transcortin-like component, also produces an inhibition of VIP-induced cAMP accumulation. Corticosterone also inhibits VIP-induced cAMP production but at concentrations higher than those of dexamethasone. In contrast, aldosterone, progesterone, estradiol, and testosterone have no effect. These results demonstrate that, in normal rat pituitary cells in culture, glucocorticoids at physiological concentrations rapidly inhibit the cAMP production and prolactin release induced by VIP by acting through specific glucocorticoid receptors.
Animals↗
In vitro autoradiographic localization of vasoactive intestinal peptide (VIP) binding sites in the rat central nervous system.
This paper describes the autoradiographic distribution of VIP binding sites in the rat central nervous system using monoiodinated 125I-labeled VIP. High densities of VIP binding sites are observed in the granular layer of the dorsal dentate gyrus of the hippocampus, the basolateral amygdaloid nucleus, the dorsolateral and median geniculate nuclei of the thalamus as well as in the ventral part of the hypothalamic dorsomedial nucleus.
Animals↗
Autoradiographic localization of a non-reducible somatostatin analog (125I-CGP 23996) binding sites in the rat brain: comparison with membrane binding.
The regional distribution of somatostatin binding sites in the rat brain was determined by quantitative autoradiography, using 125I-CGP 23996, a non-reducible somatostatin analog. In preliminary experiments, kinetic properties of 125I-CGP 23996 binding to rat brain membranes and slide mounted frozen brain sections were compared and found similar. In addition, distribution of 125I-CGP 23996 and 125I-N-Tyr-SRIF14 binding sites on membrane prepared from 10 different rat brain structures were closely correlated (r = 0.91, 2 p less than 0.01), indicating that the non-reducible analog recognizes the same binding site as the Tyr-extended native peptide. Highest levels of 125I-CGP 23996 binding sites were found in anterior temporal, frontal and cingular cortex as well as hippocampus. Moderate levels were found in the remaining part of the limbic system including amygdala, olfactory tubercles and bed nucleus of the stria terminalis. In the brain stem, nuclei involved in the auditory system such as the ventral cochlear nucleus and the superior olive nucleus, contained high levels of 125I-CGP 23996 binding sites. The distribution of 125I-CGP 23996 binding sites roughly correlated with that of the endogenous peptide in most structures, except in the mediobasal hypothalamus.
Amygdala↗
Enhanced targeting specificity to tumor cells by simultaneous recognition of two antigens.
Radioimmunotherapy recently afforded convincing results for B-cell non-Hodgkin's lymphoma treatment with antibody specific for B-cell differentiation antigens. High doses of unlabeled or labeled antibodies are necessary to saturate specific sites on normal B-cells. We thus developed a new targeting strategy, taking advantage of dual binding cooperativity, to enhance the specificity of the radioactive uptake by tumor cells. This approach was evaluated using human Burkitt lymphoma cells (Ramos) which express both CD10 and CD20 antigens. Most normal cells express at most one of these two differentiation antigens but many hematological tumors, including most human B type acute lymphoblastic leukemia cells, express both. Cells pretargeted with two bispecific antibodies, one recognizing CD10 and a histamine derivative (HSG), the other recognizing CD20 and the DTPA-indium complex, bind cooperatively radiolabeled mixed-haptens (DTPA-HSG). Increased binding (about 5-fold compared to binding to only one of CD10 or CD20 antigens) is observed at 37 degrees C, demonstrating the feasibility of the technique. This binding enhancement is a slow process, not observed at 4 degrees C. Such a binding enhancement will increase specificity for targeting isotopes to double antigen positive tumor cells compared to nontumor tissue cells bearing only one of them. This approach might be used to increase tumor irradiation with minimal irradiation of normal cells.
Animals↗
[Autoradiographic localization and characterization of the ocular binding sites of the VIP (vasoactive intestinal peptide) in albino rats and rabbits].
Despite its name, Vasoactive Intestinal Peptide (VIP), a 28-amino acid peptide, is widely distributed in the eye where it is thought to play a physiological role, particularly in aqueous humor dynamics or retinal neurotransmission. Localization and pharmacological properties of VIP binding sites were investigated in eyes from albino rabbit and rat using an in vitro autoradiographic method. 125I-VIP was used as ligand and unlabelled VIP was used to displace labelled VIP. Autoradiograms were generated by apposing the slides to 3H-Ultrofilm or autoradiographic emulsion and analysed using an image analysis system. Specific binding represented about 85% of total binding. Kinetic studies showed that equilibrium was reached after 140 min incubation at room temperature. Biochemical investigations demonstrated that 125I-VIP bound to a population of sites with high affinity (Kd = 2.95 +/- 0.5 nM). Inhibition of 125I-VIP binding with VIP and related peptide gave a rank order of potency: VIP greater than peptide histidine isoleucine greater than secretin greater than human growth hormone-releasing factor, glucagon, VIP1-14, VIP14-28. In both species, specific binding were found in conjunctiva, iris, ciliary processes, choroid and retina. Quantitative analysis of autoradiograms revealed that the highest densities of binding sites were localized in the ciliary epithelium in rabbits and in the inner retina in rats.
Animals↗