Different postnatal ontogeny of two [3H]neurotensin binding sites in rat brain

doi:10.1016/0006-8993(87)90398-2

Brain Research

Volume 408, Issues 1–2, 7 April 1987, Pages 326-328

https://doi.org/10.1016/0006-8993(87)90398-2 Get rights and content

Abstract

Two distinct neurotensin binding sites have been identified in rat brain: the NT₁-acceptor site (levocabastine-sensitive) and the NT₂-receptor site. In rat forebrain, NT₂-receptor were present at birth, revealed a maximal level (13.8 fmol/mg tissue) on day 10 of postnatal life but a much lower plateau (3.0 fmol/mg tissue) in adult rats. NT₁-acceptors were not detected before day 10 and became maximal at day 30. It is suggested that the loss of NT₂-receptor sites during the postnatal development may be the expression of the regression of a transient redundancy of neuronal connections.

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Cited by (58)

Synthesis and binding characteristics of [<sup>3</sup>H]neuromedin N, a NTS2 receptor ligand
2016, Neuropeptides
Citation Excerpt :
Previous reports indicate that the Zn metallo-exopeptidase aminopeptidase M is largely responsible for NN degradation in vitro and in vivo in the gastrointestinal tract (Barelli et al. 1995; Kitabgi 2006). Two types of neurotensin receptors, belonging to the seven transmembrane domain GPCRs, and previously distinguished by their affinity for neurotensin and their sensitivity to the antihistaminic drug levocabastine (Schotte and Laduron 1987), were cloned. The high-affinity, levocabastine-insensitive, neurotensin receptor NTS1 was the first to be cloned (Vita et al. 1993), followed in 1996 by the low-affinity levocabastine-sensitive neurotensin receptor NTS2 (Chalon et al. 1996).
Neurotensin (NT) and its analog neuromedin N (NN) are formed by the processing of a common precursor in mammalian brain tissue and intestines. The biological effects mediated by NT and NN (e.g. analgesia, hypothermia) result from the interaction with G protein-coupled receptors.
The goal of this study consisted of the synthesis and radiolabeling of NN, as well as the determination of the binding characteristics of [³H]NN and G protein activation by the cold ligand. In homologous displacement studies a weak affinity was determined for NN, with IC₅₀ values of 454 nM in rat brain and 425 nM in rat spinal cord membranes. In saturation binding experiments the K_d value proved to be 264.8 ± 30.18 nM, while the B_max value corresponded to 3.8 ± 0.2 pmol/mg protein in rat brain membranes. The specific binding of [³H]NN was saturable, interacting with a single set of homogenous binding sites. In sodium sensitivity experiments, a very weak inhibitory effect of Na⁺ ions was observed on the binding of [³H]NN, resulting in an IC₅₀ of 150.6 mM. In [³⁵S]GTPγS binding experiments the E_max value was 112.3 ± 1.4% in rat brain and 112.9 ± 2.4% in rat spinal cord membranes and EC₅₀ values of 0.7 nM and 0.79 nM were determined, respectively. NN showed moderate agonist activities in stimulating G proteins. The stimulatory effect of NN could be maximally inhibited via use of the NTS2 receptor antagonist levocabastine, but not by the opioid receptor specific antagonist naloxone, nor by the NTS1 antagonist SR48692. These observations allow us to conclude that [³H]NN labels NTS2 receptors in rat brain membranes.
Pharmacology and functional properties of NTS2 neurotensin receptors in cerebellar granule cells
2002, Journal of Biological Chemistry
The binding and signaling properties of neuronal NTS2 neurotensin (NT) receptors were examined in cultured rat cerebellar granule cells. As shown by reverse transcription-PCR, receptor autoradiography, and confocal microscopic localization of fluorescent NT, these cells selectively express the NTS2 receptor subtype. Accordingly, a single apparent class of¹²⁵I-NT-binding sites, with an affinity of 3.1 nm, was detected in cerebellar granule cell cultures. This binding was competed for with high affinity (IC₅₀ = 5.7 nm) by the NTS2 ligand levocabastine and with low affinity (IC₅₀ = 203 nm) by the NTS1 antagonist SR48692. Hypertonic acid stripping of surface-bound ligand and hyperosmolar sucrose treatment revealed that 64% of specifically bound¹²⁵I-NT was internalized at equilibrium via a clathrin-dependent pathway. In cells loaded with the Ca²⁺-sensitive fluorescent dye Fluo4, SR48692, but neither NT nor levocabastine, triggered a marked increase in cytosolic [Ca²⁺]_i. By contrast, both NT and levocabastine, but not SR48692, induced a sustained (>60 min) activation of the mitogen-activated protein kinases, p42/p44, indicating functional coupling of NTS2 receptors. Complementary experiments carried out on synaptosomes from adult rat cerebellum demonstrated the presence of presynaptic NTS2 receptors. However, in contrast to perikaryal NTS2 sites, these presynaptic receptors did not internalize in response to NT stimulation. Taken together, the present results demonstrate that NTS2 receptors are present both presynaptically and postsynaptically in central neurons and that NT and levocabastine act as agonists on these receptors.
Chapter VI Neurotensin receptors in the central nervous system
2002, Handbook of Chemical Neuroanatomy
This chapter reviews the discovery of NT and briefly discusses its synthesis, degradation, distribution, and biological effects in the CNS. It focuses on the characterization and distribution of NT receptors and of their mRNA in adult and developing mammalian brain. Like many other neuropeptides, the tridecapeptide neurotensin (NT) is a messenger of intercellular communication working as a neurotransmitter or neuromodulator in the brain and as a local hormone in the periphery. Several pharmacological, morphological, and neurochemical data suggest that NT is involved in many physiological processes in the central nervous system (CNS), as well as in the gastrointestinal tract. Both central and peripheral modes of action of NT imply as a first step the recognition of the peptide by a specific receptor located on the plasma membrane of the target cell. The activated ligand-receptor complex then transduces the extracellular signal inside the cell.
Mouse neurotensin receptor 2 gene (Ntsr2): Genomic organization, transcriptional regulation and genetic mapping on chromosome 12
2001, Molecular Brain Research
We earlier isolated and characterized the mouse neurotensin receptor 1 (Ntsr1) gene and developed Ntsr1 null mice. In the present study, we isolated the mouse neurotensin receptor 2 gene (Ntsr2) and characterized the structure. The gene fragments available to us have 1.1 kb of 5′ upstream promoter region and a 7 kb coding region composed of four exons. Transcription initiation sites, determined by primer extension analysis, are located at 286 and 303 bp upstream from initiation of the ATG codon. The promoter region contains a TATA-like box, a typical CAAT box and putative GATA-2, CREB, Oct-2 and Ikarous 2 binding elements. We also found novel splice donor–acceptor sites for alternative splicing, which could generate a short form of mRNA encoding a truncate-type receptor. In addition, we determined the chromosomal location of the Ntsr2 gene and mapped it at 6 cM from the centromere on chromosome 12.
Distribution of the neurotensin receptor NTS1 in the rat CNS studied using an amino-terminal directed antibody
2000, Neuropharmacology
The distribution of neurotensin receptor 1 immunoreactivity in the rat brain was studied using an antibody against the amino-terminal of the receptor expressed as a fusion protein with glutathione-S transferase. Affinity purified antibodies detected the fusion protein and the complete neurotensin receptor sequence expressed in Escherichia coli. The immunostaining was abolished by preabsorption with the amino-terminal fusion protein. Immunoreactive neurotensin receptor 1 immunoreactivity was detected on cell bodies and their processes in a number of CNS regions. In agreement with previous binding studies neurotensin receptor 1 immunoreactivity was particularly localised in cell bodies in the basal forebrain, nucleus basalis and substantia nigra. At the electron microscope level immunoreactivity was found both in axonal bouton and dendrites and spines in the basal forebrain indicating that neurotensin may act both pre- and post-synaptically. There were several regions such as the substantia gelatinosa, ventral caudate-putamen and the lateral reticular nucleus where the neurotensin receptor 1 positive cells had not previously been reported, indicating that distribution of this receptor is widespread.
Pharmacological, molecular and functional characterization of glial neurotensin receptors
1999, Neuroscience
The pharmacological properties, molecular identity and physiopathological regulation of neurotensin receptors expressed by central astrocytes were investigated in primary glial cultures and sections from the adult rat brain. Binding experiments carried out on astrocytes in culture revealed the presence of a single apparent class of neurotensin binding sites. These sites bound [¹²⁵I]neurotensin with an affinity (6 nM) comparable to that of the recently cloned NT2 low-affinity receptor expressed in transfected cells. The glial receptor was sensitive to the antihistamine, levocabastine, but less so than the NT2 site expressed in heterologous expression systems, suggesting the presence of an additional site or a differential coupling of the NT2 receptor in glia. Reverse transcription–polymerase chain reaction experiments demonstrated that both NT2 and NT3 neurotensin receptor sub-types were in fact expressed by cortical glial cells in culture. Confocal microscopic visualization of specifically bound fluorescent neurotensin indicated that this expression concerned only a sub-population of astrocytes in culture, in conformity with earlier reports of a heterogeneous expression of neuropeptides and their receptors by glial cells. To further investigate the functionality of NT2 receptors expressed in astrocytes, dual immunohistochemical labeling of glial fibrillary acidic protein and in situ hybridization of NT2 messenger RNA was performed on sections of normal and lesioned rat brain. In sections from normal brain, only a small subset of immunolabeled astrocytes hybridized NT2 messenger RNA. By contrast, in sections of stab-wounded rat brains, there was a marked increase in the number of NT2-hybridizing astrocytes in the surround of the lesion. Furthermore, NT2 expression within immunopositive reactive astrocytes was significantly enhanced as compared to immunolabeled glial cells in the brain of control animals.
These results indicate that NT2 receptor expression is up-regulated during astrocytic reaction, suggesting that NT2 receptors may play a role in regulating glial response to injury.

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: Part of this work was supported by a grant from the I.W.O.N.L. (Brussels, Belgium).

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Abstract

Reference (20)

Postnatal changes in glia neuron index with a comparison of methods of cell enumeration in the white rat

Prog. Brain Res.

The isolation of a new hypotensive peptide, neurotensin, from bovine hypothalami

J. Biol. Chem.

Characterization of radioimmunoassayable neurotensin in the rat; its differential distribution in the central nervous system, small intestine and stomach

J. Biol. Chem.

The regional distribution of somatostatin, substance P and neurotensin in human brain

Brain Research

Neurotensin — a status report

TINS

Neurotensin receptors in the rat striatum: lesion studies

Brain Research

Postnatal ontogeny of cholecytokinin receptors in rat brain

Brain Research

Criteria for receptor sites in binding studies

Biochem. Pharmacol.

Monoiodo-[Trp¹¹]neurotensin, a highly radioactive ligand of neurotensin receptors

J. Biol. Chem.

Ontogeny of α₁-andα₂-adrenoceptors in rat brain

Brain Research

Cited by (58)

Synthesis and binding characteristics of [<sup>3</sup>H]neuromedin N, a NTS2 receptor ligand

Pharmacology and functional properties of NTS2 neurotensin receptors in cerebellar granule cells

Chapter VI Neurotensin receptors in the central nervous system

Mouse neurotensin receptor 2 gene (Ntsr2): Genomic organization, transcriptional regulation and genetic mapping on chromosome 12

Distribution of the neurotensin receptor NTS1 in the rat CNS studied using an amino-terminal directed antibody

Pharmacological, molecular and functional characterization of glial neurotensin receptors