Elsevier

Neuroscience

Volume 94, Issue 4, November 1999, Pages 1189-1197
Neuroscience

Pharmacological, molecular and functional characterization of glial neurotensin receptors

https://doi.org/10.1016/S0306-4522(99)00354-1Get rights and content

Abstract

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.

Section snippets

Materials

Neurotensin was purchased from Peninsula Laboratories. [125I]Tyr3-NT1–13 ([125I]NT) and Nα-Bodipy-NT2–13 were prepared and purified as described previously.2., 28., 32. The expression vector pcDNA3 was from Invitrogen (San Diego, CA, U.S.A.), the vector pBluescript™ KS from Stratagene (La Jolla, CA, U.S.A.) and the vector P-target from Promega (Lyon, France). Dulbecco's modified Eagle's medium and penicillin/streptomycin were purchased from Life Technologies (Gaithersburg, MD, U.S.A.) and fetal

Binding studies

Immunohistochemical characterization of cells present in cortical glial cultures 15 days after plating revealed that virtually all viable cells were GFAP immunopositive (>95%) and could therefore be considered as astrocytes.

Incubation of cultured glial cells in the presence of increasing doses of [125I]NT for 45 min at 37°C revealed the presence of specific (i.e. displaceable) and saturable [125I]NT binding (Fig. 1). This binding was confined to the cell surface, as it was entirely strippable by

Discussion

The present study demonstrates that rat forebrain astrocytes express both NT2 and NT3 neurotensin receptor subtypes. It also shows that NT2 receptor expression is up-regulated in reactive astrocytes surrounding a stab wound of the brain, and that it may therefore play a role in inflammatory reaction and/or in neuronal regeneration.

Binding experiments carried out on astrocytes in culture demonstrated saturable specific [125I]NT binding to a single apparent population of sites. The affinity of NT

Acknowledgements

This study was supported by Grant MA-7366 from the Medical Research Council of Canada and by a fellowship to D.N., and a France–Québec exchange program to J.M. and A.B. from the Fonds de la Recherche en Santé du Québec. The technical assistance of Mariette Houle is gratefully acknowledged.

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