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.
Highlights
Neurotensin (NT)1 is a tridecapeptide documented to act as a neurotransmitter/neuromodulator in the central nervous sys
The present study reveals that NTS2 neurotensin receptors are associated with somatodendritic and terminal arbors of rat cerebellar granule cells
Radiolabeled NTS2 receptors were most conspicuous in the granule cell layer, in keeping with earlier in situ hybridization data, which demonstrated high expression of NTS2 mRNA in granule cells of mouse cerebellar cortex [10]
Summary
Neurotensin (NT) is a tridecapeptide documented to act as a neurotransmitter/neuromodulator in the central nervous sys-. In the central nervous system, NT has been shown to modulate dopamine transmission in nigrostrial and mesolimbic pathways [2] and to play a role in the regulation of pain, temperature, appetite, and pituitary hormone secretion (for review see Ref. 3). Three different subtypes of NT receptors, referred to as NTS1, NTS2, and NTS3, have been cloned (for review see Ref. 4). Most of the documented effects of NT appear to be exerted through the high affinity, NTS1 receptor, recent studies suggest that the levocabastine-sensitive, low affinity NTS2 subtype may be responsible for the mediation of the antinociceptive actions of NT [5]. Whereas the binding and internalization properties of NTS2 have been characterized in astrocytes [14], nothing is known of its pharmacological and functional properties in neurons
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