Danielle Gully

The 100-kDa neurotensin receptor is gp95/sortilin, a non-G-protein-coupled receptor

In this work, the 100-kDa neurotensin (NT) receptor previously purified from human brain by affinity chromatography (Zsürger, N., Mazella, J., and Vincent, J. P. (1994) Brain Res. 639, 245–252) was cloned from a human brain cDNA library. This cDNA encodes a 833-amino acid protein 100% identical to the recently cloned gp95/sortilin and was then designated NT3 receptor-gp95/sortilin. The N terminus of the purified protein is identical to the sequence of the purified gp95/sortilin located immediately after the furin cleavage site. The binding of iodinated NT to 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid-solubilized extracts of COS-7 cells transfected with the cloned cDNA was saturable and reversible with an affinity of 10–15 nm. The localization of the NT3 receptor-gp95/sortilin into intracellular vesicles was in agreement with previous results obtained with the purified receptor and with gp95/sortilin. Affinity labeling and binding experiments showed that the 110-kDa NT3 receptor can be partly transformed into a higher affinity (K d = 0.3 nm) 100-kDa protein receptor by cotransfection with furin. This 100-kDa NT receptor corresponded to the mature form of the receptor. The NT3/gp95/sortilin protein is the first transmembrane neuropeptide receptor that does not belong to the superfamily of G-protein-coupled receptors.

Cloning and expression of a complementary DNA encoding a high affinity human neurotensin receptor

A human neurotensin receptor (hNTR) cDNA was cloned from the colonic adenocarcinoma cell line HT29. The cloned cDNA encodes a putative peptide of 418 amino acids with 7 transmembrane domains. The amino acid sequence of the hNTR is 84% identical to the rat NTR [Neuron, 4 (1990) 847‐854]. Transfection of this cDNA into COS cells results in the expression of receptors with pharmacological properties similar to those found with HT29 cells. Northern blot analysis using the hNTR cDNA probe indicated a single transcript of 4 kb in the brain, the small intestine and blood mononuclear cells.

Peripheral biological activity of SR 27897: a new potent non-peptide antagonist of CCKA receptors

SR 27897 is a new non-peptide antagonist of CCKA receptors: 1-[[2-(4-(2-chlorophenyl)thiazol-2-yl)aminocarbonyl] indolyl] acetic acid. This compound is a potent ligand for CCKA binding sites (rat pancreatic membranes, Ki = 0.2 nM) and is highly selective (CCKB and gastrin/CCKA IC50 ratios of 800 and 5000 respectively). In vitro, it is a competitive antagonist of cholecystokinin (CCK)-stimulated amylase release in isolated rat pancreatic acini (pA2 = 7.50) and of CCK-induced guinea pig gall bladder contractions (pA2 = 9.57). In in vivo gastrointestinal models, SR 27897 confirmed the potency obtained in vitro: at 1 mg/kg (i.v.) it completely reversed the CCK-induced amylase secretion, at 3 micrograms/kg (p.o.) it antagonized by 50% the CCK-induced inhibition of gastric emptying of a charcoal meal in mice, and 72 micrograms/kg (p.o.) was the median effective dose for inhibiting CCK-induced gall bladder emptying in mice. SR 27897 was also very active (ED50 = 27 micrograms/kg p.o.) in the gall bladder emptying protocol with egg yolk as an inducer of endogenous CCK release. SR 27897 had a long-lasting action in all the experiments, with no differences between oral and intravenous routes of administration. SR 27897 was more or less effective than L-364,718, depending on the model and the species. Both compounds increased the gall bladder volume of fasting mice, but the effect of SR 27897 was 10 times lower than that of L-364,718. In summary, SR 27897 is a selective antagonist of CCKA receptors, is highly potent in animal models whatever the route of administration and has a long duration of action.(ABSTRACT TRUNCATED AT 250 WORDS)

Stable Expression of the Cloned Rat Brain Neurotensin Receptor into Fibroblasts: Binding Properties, Photoaffinity Labeling, Transduction Mechanisms, and Internalization

Abstract: The study of the pharmacological, biochemical, and transduction properties of the cloned rat brain neurotensin receptor was carried out in thymidine kinase mutant fibroblasts stably transfected with the receptor cDNA. The interaction of neurotensin with transfected fibroblasts leads to a concentration‐dependent stimulation of phosphatidylinositol hydrolysis and intracellular calcium. These effects are totally inhibited by the nonpeptide neurotensin antagonist SR48692. By contrast, this receptor remains unable to modulate intracellular levels of cyclic nucleotides. The transfected neurotensin receptor can be solubilized in an active form by digitonin with an identical pharmacological profile, whereas the detergent 3‐[(3‐cholamidopropyl)dimethylammonio]‐1‐propane‐sulfonic acid is unable to solubilize the binding activity. The binding of iodinated neurotensin to transfected fibroblasts bearing the cloned receptor remains partly undissociated even after an acid washing step, indicating that the transfected neurotensin receptor retains the capacity to be internalized according to a temperature‐dependent mechanism. Indeed, the sequestration of the neurotensin‐receptor complex can be blocked by phenylarsine oxide. Finally, photoaffinity labeling experiments reveal that the cloned rat brain neurotensin receptor is expressed under two forms with molecular masses of 50 and 60 kDa. Labeling and internalization of these two proteins are totally blocked by the neurotensin antagonist SR48692.

Turning behavior induced by intrastriatal injection of neurotensin in mice: sensitivity to non-peptide neurotensin antagonists

The intrastriatal injection of neurotensin (10 pg/mouse) elicited vigorous contralateral rotations which were not affected by disruption of dopaminergic transmission using 6-OHDA lesion of the striatum or systemic administration of spiroperidol (0.03 mg/kg). SR 48692, a selective non-peptide antagonist of neurotensin receptor, produced the following pattern of changes: a significant antagonism of rotations was observed at 0.04 and 0.08 mg/kg i.p. followed by a reinstatement of rotations at 0.16–0.64 mg/kg (at higher doses, a second antagonism occurred that lacked stereo selectivity). The reinstatement of rotations observed at 0.16 and 0.32 mg/kg of SR 48692 was abolished by spiroperidol and 6-OHDA lesions, suggesting the role of dopamine regulatory mechanisms.

Neurotensin receptor interaction with dopaminergic systems in the guinea-pig brain shown by neurotensin receptor antagonists

Neurotensin has been suggested to be involved in neurological and mental disorders associated with altered dopaminergic transmission. The lack of a potent neurotensin receptor antagonist had prevented us from studying the real physiological implication of this peptide in brain function. We thus recently developed such a non-peptide neurotensin receptor antagonist, SR 48692, (2-(1-(7-chloroquinolin-4-yl)-5-(2,6-dimethoxyphenyl)-1H-pyrazole- 3-carbonyl)amino)-adamantane-2-carboxylic acid), which appeared to be potent in various central and peripheral preparations. In the present study, we tested the pharmacological properties of SR 48692 and of two optically synthetic analogs of this compound on neurotensin binding to both adult guinea-pig brain membrane homogenates and coronal brain sections, as well as on neurotensin stimulation of the K(+)-evoked release of [3H]dopamine in guinea-pig striatal slices. Our results demonstrated that (1) high-affinity neurotensin binding sites are present in the guinea-pig brain in regions rich in both dopamine cell bodies and terminals; (2) the binding of neurotensin is inhibited by SR 48692 and its related S(+) active analog, SR 48527, with IC50 values in the nM range and (3) the non-peptide antagonist has no agonist effect but antagonizes neurotensin-induced [3H]dopamine release from guinea-pig striatal nerve terminals.

Neurobehavioural effects of SR 27897, a selective cholecystokinin type A (CCK-A) receptor antagonist

SummaryThe activity of SR 27897, a potent and selective CCK-A vs CCK-B receptor antagonist (Ki = 0.2 nM on guinea-pig pancreas vs 2000 nM on rat brain) was studied on behavioural, electrophysiological and biochemical effects induced by peripheral or central injection of CCK-8S. For comparative purposes, devazepide, a reference CCK-A receptor antagonist, was investigated in these same models. CCK-induced hypophagia and CCK-induced hypolocomotion in rats, two behavioural changes associated with the stimulation of peripheral CCK-A receptors, were dose-dependently antagonized by SR 27897 (ED50 = 0.003 and 0.002 mg/kg i.p., respectively) and devazepide (ED50 = 0.02 and 0.1 mg/kg i.p., respectively). CCK-induced decrease of cerebellar cGMP levels in mice was also reduced by SR 27897 (ED50 = 0.013 mg/kg) and by devazepide (0.084 mg/kg). The CCK-induced turning behaviour after intrastriatal injection in mice, and the potentiation of the rate suppressant activity of apomorphine on rat DA neurons, were blocked by higher doses of SR 27897 and devazepide, consistent with the probable central origin of these effects. The respective ED50s were 0.2 mg/kg i.p. for SR 27897 and 4.9 mg/kg i.p. for devazepide in the former model, while the respective minimal effective doses were 1.25 and 5 mg/kg i.p. in the latter test. In most tests the i.p./p.o. ratio for SR 27897 was near unity, suggesting a high oral bioavailability of the compound. Taken together, these findings support the notion that SR 27897 behaves as a potent CCK-A antagonist able to cross the blood brain barrier.

SR 48692 inhibits neurotensin-induced [3H]dopamine release in rat striatal slices and mesencephalic cultures.

In rat striatal slices, the increase (114 +/- 11%) in K(+)-evoked [3H]dopamine release induced by neurotensin (10 nM) was antagonized by 2-[(1-(7-chloro-4-quinolinyl)-5-(2,6-dimethoxyphenyl)pyrazol-3-yl) carboxylamino]tricyclo(3.3.1.1.3.7)decan-2-carboxylic acid (SR 48692, IC50 = 1.2 +/- 0.11 nM). SR 48692 (100 nM) also suppressed the neurotensin (10 nM)-induced increase (47%) in K(+)-evoked [3H]dopamine release in primary cultures of fetal rat mesencephalic cells. These results further characterize SR 48692 as a potent antagonist of neurotensin receptors in the rat.

Neuropharmacological profile of non-peptide neurotensin antagonists

Summary— Neurotensin, an endogenous peptide widely distributed throughout the brain, fulfils neurotransmitter criteria. When administered centrally, neurotensin induces various effects and modulates the activity of the mesolimbic dopamine system. It antagonizes the behavioural action of dopamine in a manner similar, but not identical, to antipsychotic drugs. Neurotensin is even considered to be an endogenous neuroleptic. In fact, microinjection of neurotensin elicits different effects depending on both the dose and the cerebral structures into which the injection is made. Our work on the development of orally‐active neurotensin antagonists has led to the identification of SR 48692, the first non‐peptide antagonist of the neurotensin receptor, and some analogues. This small molecule reveals a surprising neuropharmacological profile. It antagonizes turning behaviour induced in mice and rats (after striatal or ventral tegmental area administration of neurotensin, respectively), hypolocomotion induced by intracerebroventricular injection of neurotensin in rats, and reverses the inhibitory effect of neurotensin (nucleus accumbens injection) on amphetamine‐induced hyperlocomotion in rats. However, SR 48692 cannot reverse either dopamine release in the nucleus accumbens evoked by neurotensin injection in ventral tegmental area, or hypothermia and analgesia induced by intracerebroventricular injection of neurotensin. As direct and indirect dopamine agonists have been reported to promote neurotensin release in the cortex, behavioural studies were performed using injection of apomorphine. In these experiments, SR 48692 inhibited only turning and yawning. It did not antagonize other apomorphine‐dependent effects such as climbing, hypothermia, hypo‐ or hyperlocomotion, penile erection and stereotypies. All together, these data raise the question of the existence of neurotensin receptor subtypes and confirm that the nature of neurotensin and dopamine interactions depends on the brain structures considered.

Characterization of the effect of SR48692 on inositol monophosphate, cyclic GMP and cyclic AMP responses linked to neurotensin receptor activation in neuronal and non‐neuronal cells

1 Neurotensin stimulated inositol monophosphate (IP1) formation in both human colonic carcinoma HT29 cells and in mouse neuroblastoma N1E115 cells with EC50 values of 3.5 ± 0.5 nM (n = 4) and 0.46±0.02 nM (n = 3), respectively. Neurotensin also stimulated cyclic GMP production with an EC50 of 0.47±1.2 nM and inhibited cyclic AMP accumulation induced by forskolin (0.5 μm) with an IC50 of 1.33 + 1.5 nM (n = 3) on the N1E115 cell line 2 The competitive antagonism by the non‐peptide neurotensin receptor antagonist, SR48692 of neurotensin‐induced IP1 formation revealed pA2 values of 8.7 ±0.2 (n = 3) for HT29 and 10.1 ±0.2 (n = 3) for N1E115 cells. SR48692 also antagonized the cyclic GMP and cyclic AMP responses induced by neurotensin in the N1E115 cell line with pA2 values of 10.7±0.7 (n = 3) and 9.8±0.3 (n = 3), respectively 3 In CHO cells transfected with the rat neurotensin receptor, neurotensin stimulated IP1 and cyclic AMP formation with EC50 values of 3.0±0.5 nM (n = 3) and 72.2 ±20.7 nM (n = 3), respectively. Both effects were antagonized by SR48692, giving pA2 values of 8.4 ±0.1 (n = 3) for IP1 and 7.2 ±0.4 (n = 3) for cyclic AMP responses 4 Radioligand binding experiments, performed with [125I]‐neurotensin (0.2 nM), yielded IC50 values of 15.3 nM (n = 2) and 20.4 nM (n = 2) for SR48692 versus neurotensin receptor binding sites labelled in HT29 and N1E115 cells, respectively 5 In conclusion, SR48692 appears to be a potent, species‐independent antagonist of the signal transduction events triggered by neurotensin receptor activation in both neuronal and non‐neuronal cell systems.