Autoradiographic and electrophysiological evidence for the existence of neurotensin receptors on cultured astrocytes

doi:10.1016/0306-4522(95)00005-4

Neuroscience

Volume 66, Issue 3, June 1995, Pages 627-633

https://doi.org/10.1016/0306-4522(95)00005-4 Get rights and content

Abstract

By means of autoradiography we have studied the cellular localization of binding sites for [³H]neurotensin and its nonpeptide receptor antagonist [³H]SR-48692 in explant cultures of rat neocortex, striatum, brain stem and spinal cord. Binding sites for the peptide and its antagonist were observed on a great number of astrocytes in all CNS regions studied. Simultaneous staining of the cultures with a monoclonal antibody against glial fibrillary acidic protein has shown that the labelled cells in the outgrowth zone of the cultures were glial fibrillary acidic protein-positive and could therefore be identified as astrocytes. In addition to astrocytes, many neurons and outgrowing nerve fibres were labelled by the radioligands. Binding of [³H]neurotensin and [³H]SR-48692 (10⁻⁸M) to neurons and glial cells was markedly reduced or inhibited by the unlabelled compounds at high concentration (10⁻⁶M), suggesting “specific” binding of the radioligands. Electrophysiological studies have shown that addition of neurotensin to the bathing solution caused a hyperpolarization of the majority of astrocytes tested. There was a dose-response relationship between the magnitude of the hyperpolarization and the concentration of the peptide (10⁻¹⁰−10⁻⁷M); 10⁻¹⁰M being the threshold concentration. The specificity of the action of neurotensin was confirmed by the selective nonpeptide neurotensin receptor antagonist SR-48692 which reversibly blocked or markedly reduced the hyperpolarization by the peptide on all astrocytes tested.

Our electrophysiological findings together with our autoradiographic data provide strong evidence for the presence of specific and functional neurotensin receptors on astrocytes.

Reference (37)

CarrawayR.E. et al.
The isolation of a new hypotensive peptide, neurotensin, from bovine hypothalami
J. biol. Chem.
(1973)
CastelM.N. et al.
Immunohistochemical evidence for a neurotensin striatonigral pathway in the rat brain
Neuroscience
(1993)
DanaC. et al.
Characterization of neurotensin binding sites on rat mesencephalic cells in primary culture
Dev. Brain Res.
(1991)
ErvinG.N. et al.
Interactions of neurotensin with dopamine-containing neurons in the central nervous system
Prog. Neuro-Psych. Biol. Psych.
(1988)
HosliE. et al.
Receptors for neurotransmitters on astrocytes in the mammalian central nervous system
Prog. Neurobiol.
(1993)
HosliE. et al.
Binding of cholecystokinin, bombesin and muscarine to neurons and astrocytes in explant cultures of rat central nervous system: Autoradiographic and immunohistochemical studies
Neuroscience
(1994)
HosliL. et al.
Electrophysiological evidence for receptors for vasoactive intestinal peptide and angiotensin II on astrocytes of cultured rat central nervous system
Neurosci. Lett.
(1989)
KalivasP.W.
Interactions between neuropeptides and dopamine neurons in the ventromedial mesencephalon
Neurosci. Biobehav. Rev.
(1985)
MatthewE.
Neuropeptides in dissociated cell cultures of mammalian spinal cord and dorsal root ganglion
Int. J. devl Neurosci.
(1993)
MercuriN.B. et al.
Neurotensin induces an inward current in rat mesencephalic dopaminergic neurons
Neurosci. Lett.
(1993)

MoyseE. et al.

Distribution of neurotensin binding sites in rat brain: a light microscopic radioautographic study using monoiodo [¹²⁵I]Tyr₃-neurotensin

Neuroscience

(1987)

PinnockR.D.

Neurotensin depolarizes substantia nigra dopamine neurones

Brain Res.

(1985)

QuirionR. et al.

Autoradiographic distribution of [³H]neurotensin receptors in rat brain: Visualization by tritium-sensitive film

Peptides

(1982)

QuirionR.

Interactions between neurotensin and dopamine in the brain: An overview

Peptides

(1983)

QuirionR. et al.

Comparative localization of neurotensin receptors on nigrostriatal and mesolimbic dopaminergic terminals

Brain Res.

(1985)

ReinerA. et al.

Co-occurrence of γ-aminobutyric acid, parvalbumin and the neurotensin-related neuropeptide LANT6 in pallidal nigral and striatal neurons in pigeons and monkeys

Brain Res.

(1993)

SeutinV. et al.

Electrophysiological effects of neurotensin on dopaminergic neurones of the ventral tegmental area of the rat in vitro

Neuropharmacology

(1989)

SteinbergR. et al.

SR 48692, a non-peptide neurotensin receptor antagonist differentially affects neurotensin-induced behaviour and changes in dopaminergic transmission

Neuroscience

(1994)

Cited by (20)

Astrocytic receptors and second messenger systems
2003, Advances in Molecular and Cell Biology
Citation Excerpt :
Two different receptors for VIP have been identified: one with a low-affinity binding site coupled to activation of adenylate cyclase (Gozes et al., 1991), and the other with a high-affinity site, which is coupled to IP3 generation and changes in [Ca2+]i (Fatatis et al., 1994). Receptors for neurotensin and neuropeptide Y (NPY) have been identified on the basis of ligand binding and electrophysiology (Gimpl et al., 1993; Hösli et al., 1995). Two subtypes of bradykinin receptors, B1 and B2, have been described.
This chapter reviews that glial cells support neuronal activity and create opportunities for the dynamics and plasticity of the central nervous system (CNS). After injury, metabolic disturbance or toxic influence, and in degenerative disease, glial cell reactions protect neurons and facilitate rebuilding. It explores that astrocytes is the most numerous glial cells in the CNS and express a large number of receptors for neurotransmitters, peptides, purines, cytokines, and other neuroactive substances; many of them previously thought to be present only on neurons. These astrocytic receptors are coupled to G-proteins or ion channels, to intracellular protein kinases, or associated with mitochondria or cell nuclei. Even if most evidence for the existence of this extensive repertoire of functional astrocytic receptors comes from in vitro studies, convincing studies have demonstrated the presence of astrocytic receptor groups also in the intact nervous system. The chapter also discusses that the astrocytic receptors and the signaling systems, with which they are associated, set the stage for extensive neuronal–glial signaling that in turn is essential for neuroplasticity both in the intact nervous system, and after trauma and in degenerative disease. Pharmacological manipulation of astrocytic receptor systems might provide a new strategy to reinforce the reparative processes within the CNS after injury or in disease.
Nigral neurotensin receptor regulation of nigral glutamate and nigroventral thalamic GABA transmission: A dual-probe microdialysis study in intact conscious rat brain
2001, Neuroscience
Dual-probe microdialysis in the awake rat was employed to investigate the effects of intranigral perfusion with the tridecapeptide neurotensin on local dialysate glutamate and GABA levels in the substantia nigra pars reticulata and on dialysate GABA levels in the ventral thalamus. Intranigral neurotensin (10–300 nM, 60 min) dose-dependently increased (+29±3% and +46±3% vs basal for the 100 and 300 nM concentrations, respectively) local dialysate glutamate levels, while the highest 300 nM concentration of the peptide exerted a long-lasting and prolonged reduction in both local and ventral thalamic (−20±4% and −22±2%, respectively) GABA levels. Intranigral perfusion with the inactive neurotensin fragment neurotensin(1–7) (10–300 nM, 60 min) was without effect. Furthermore, the non-peptide neurotensin receptor antagonist SR 48692 (0.2 mg/kg) and tetrodotoxin (1 μM) fully counteracted the intranigral neurotensin (300 nM)-induced increase in local glutamate. SR 48692 (0.2 mg/kg) also counteracted the decreases in nigral and ventral thalamic GABA release induced by the peptide. In addition, intranigral perfusion with the dopamine D₂ receptor antagonist raclopride (1 μM) fully antagonized the neurotensin (300 nM)-induced decreases in nigral and ventral thalamic GABA levels.
The ability of nigral neurotensin receptor activation to differently influence glutamate and GABA levels, whereby it increases nigral glutamate and decreases both nigral and ventral thalamic GABA levels, suggests the involvement of neurotensin receptor in the regulation of basal ganglia output at the level of the nigra.
Neurotensin regulates intracellular calcium in ventral tegmental area astrocytes: Evidence for the involvement of multiple receptors
2000, Neuroscience
Recent evidence suggests that some types of neurotensin receptors may be expressed by astrocytes. In order to explore the function of neurotensin receptors in astrocytes, the effect of a neurotensin receptor agonist, neurotensin(8–13), on intracellular Ca²⁺ dynamics in mixed neuronal/glial cultures prepared from rat ventral tegmental area was examined. It was found that neurotensin(8–13) induces a long-lasting rise in intracellular Ca²⁺ concentration in a subset of glial fibrilary acidic protein-positive glial cells. This response displays extensive desensitization and appears to implicate both intracellular and extracellular Ca²⁺ sources. In the absence of extracellular Ca²⁺, neurotensin(8–13) evokes only a short-lasting rise in intracellular Ca²⁺. The neurotensin-evoked intracellular Ca²⁺ accumulation is blocked by the phospholipase C inhibitor U73122 and by thapsigargin, suggesting that it is initiated by release of Ca²⁺ from an inositol triphosphate-dependent store. The Ca²⁺-mobilizing action of neurotensin(8–13) in astrocytes is dependent on at least two receptors, because the response is blocked in part only by SR48692, a type 1 neurotensin receptor antagonist, and is blocked completely by SR142948A, a novel neurotensin receptor antagonist. The finding that the type 2 neurotensin receptor agonist levocabastine fails to mimic or alter the effects of neurotensin(8–13) on intracellular Ca²⁺ makes it unlikely that the type 2 neurotensin receptor is involved.
In summary, these results show that functional neurotensin receptors are present in cultured ventral tegmental area astrocytes and that their activation induces a highly desensitizing rise in intracellular Ca²⁺. The pharmacological profile of this response suggests that a type 1 neurotensin receptor is involved but that another, possibly novel, non-type 2 neurotensin receptor is also implicated. If present in vivo, such signalling could be involved in some of the physiological actions of neurotensin.
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.
Cellular aspects of trophic actions in the nervous system
1999, International Review of Cytology
During the past three decades the number of molecules exhibiting trophic actions in the brain has increased drastically. These molecules promote and/or control proliferation, differentiation, migration, and survival (sometimes even the death) of their target cells. In this review a comprehensive overview of small diffusible factors showing trophic actions in the central nervous system (CNS) is given. The factors discussed are neurotrophins, epidermal growth factor, fibroblast growth factor, platelet-derived growth factor, insulin-like growth factors, ciliary neurotrophic factor and related molecules, glial-derived growth factor and related molecules, transforming growth factor-β and related molecules, neurotransmitters, and hormones. All factors are discussed with respect to their trophic actions, their expression patterns in the brain, and molecular aspects of their receptors and intracellular signaling pathways. It becomes evident that there does not exist “the” trophic factor in the CNS but rather a multitude of them interacting with each other in a complicated network of trophic actions forming and maintaining the adult nervous system.
Peptide receptors on astrocytes
1998, Frontiers in Neuroendocrinology
In recent years, it has become apparent that astrocytes (at leastin vitro) harbor functional receptors to almost all possible neurotransmitters (with the potential noticeable exception of acetylcholine nicotinic receptors). Peptides are no exception, since receptors to all neuropeptides known to be produced in the CNS have been found on cultured astrocytes, and the presence of many of these has been confirmed on astrocytesin vivo.A variety of methodologies have been used to detect peptide receptors on astrocytes, as summarized in the current review. Special emphasis is also put on the possible roles that peptides may play in the regulation of astrocyte functions. These include proliferation, morphology, release of eicosanoids and arachidonic acid, induction of calcium transients and calcium waves, and control of internal pH, glucose uptake, glycogen metabolism, and gap junctional conductance. Recent data concerning the effects of natriuretic peptides on astrocytes are reviewed, and why these peptides may constitute priviledged tools to test the effects of peptides on astrocyte–neuron interactions is also discussed.

View all citing articles on Scopus

View full text

Autoradiographic and electrophysiological evidence for the existence of neurotensin receptors on cultured astrocytes

Abstract

J. biol. Chem.

Neuroscience

Dev. Brain Res.

Prog. Neuro-Psych. Biol. Psych.

Prog. Neurobiol.

Neuroscience

Neurosci. Lett.

Neurosci. Biobehav. Rev.

Int. J. devl Neurosci.

Neurosci. Lett.

Neuroscience

Brain Res.

Peptides

Peptides

Brain Res.

Brain Res.

Neuropharmacology

Neuroscience