title: Pharmacology and functional properties of NTS2 neurotensin receptors in cerebellar granule cells.
abstract: 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 of (125)I-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 (IC(50) = 5.7 nm) by the NTS2 ligand levocabastine and with low affinity (IC(50) = 203 nm) by the NTS1 antagonist SR48692. Hypertonic acid stripping of surface-bound ligand and hyperosmolar sucrose treatment revealed that 64% of specifically bound (125)I-NT was internalized at equilibrium via a clathrin-dependent pathway. In cells loaded with the Ca(2+)-sensitive fluorescent dye Fluo4, SR48692, but neither NT nor levocabastine, triggered a marked increase in cytosolic [Ca(2+)](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.
authors.affiliation: Department of Neurology and Neurosurgery, Montreal Neurological Institute, McGill University, Montreal, Quebec H3A 2B4, Canada.
authors.forename: Philippe
authors.initials: P
authors.lastname: Sarret
authors.forename: Louis
authors.initials: L
authors.lastname: Gendron
authors.forename: Peter
authors.initials: P
authors.lastname: Kilian
authors.forename: Ha Minh Ky
authors.initials: HM
authors.lastname: Nguyen
authors.forename: Nicole
authors.initials: N
authors.lastname: Gallo-Payet
authors.forename: Marcel-Daniel
authors.initials: MD
authors.lastname: Payet
authors.forename: Alain
authors.initials: A
authors.lastname: Beaudet
mesh_terms: D000814:Aniline Compounds / Q000494:pharmacology; D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology; D002531:Cerebellum / Q000166:cytology*; D002966:Clathrin / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D005456:Fluorescent Dyes / Q000494:pharmacology; D006634:Histamine H1 Antagonists / Q000494:pharmacology; D020128:Inhibitory Concentration 50; D008297:Male; D018613:Microscopy, Confocal; D010880:Piperidines / Q000494:pharmacology; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D011720:Pyrazoles / Q000494:pharmacology; D011804:Quinolines / Q000494:pharmacology; D012313:RNA / Q000378:metabolism; D051381:Rats; D020318:Rats, Long-Evans; D017207:Rats, Sprague-Dawley; D018028:Receptors, Neurotensin / Q000037:antagonists & inhibitors / Q000378:metabolism*; D017661:Receptors, Presynaptic / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013997:Time Factors; D014966:Xanthenes / Q000494:pharmacology
chemical_list: D000814:Aniline Compounds; D002966:Clathrin; C409648:Fluo 4; D005456:Fluorescent Dyes; D006634:Histamine H1 Antagonists; D010880:Piperidines; D011720:Pyrazoles; D011804:Quinolines; D018028:Receptors, Neurotensin; D017661:Receptors, Presynaptic; D014966:Xanthenes; C079087:SR 48692; D012313:RNA; C047340:levocabastine; D002118:Calcium