Pharmacological, molecular and functional characterization of glial neurotensin receptors
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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