Neurotensin In Mice Research Articles

Neurotensin contributes to cholestatic liver disease potentially modulating matrix metalloprotease-7

The diagnosis and treatment of biliary atresia pose challenges due to the absence of reliable biomarkers and limited understanding of its etiology. The plasma and liver of patients with biliary atresia exhibit elevated levels of neurotensin. To investigate the specific role of neurotensin in the progression of biliary atresia, the patient's liver pathological section was employed. Biliary organoids, cultured biliary cells, and a mouse model were employed to elucidate both the potential diagnostic significance of neurotensin and its underlying mechanistic pathway. In patients’ blood, the levels of neurotensin were positively correlated with matrix metalloprotease-7, interleukin-8, and liver function enzymes. Neurotensin and neurotensin receptors were mainly expressed in the intrahepatic biliary cells and were stimulated by bile acids. Neurotensin suppressed the growth and increased expression of matrix metalloprotease-7 in biliary organoids. Neurotensin inhibited mitochondrial respiration, oxidative phosphorylation, and attenuated the activation of calmodulin-dependent kinase kinase 2-adenosine monophosphate-activated protein kinase (CaMKK2-AMPK) signaling in cultured biliary cells. The stimulation of neurotensin in mice and cultured cholangiocytes resulted in the upregulation of matrix metalloprotease-7 expression through binding to its receptors, namely neurotensin receptors 1/3, thereby attenuating the activation of the CaMKK2-AMPK pathway. In conclusion, these findings revealed the changes of neurotensin in patients with cholestatic liver disease and its mechanism in the progression of the disease, providing a new understanding of the complex mechanism of hepatobiliary injury in children with biliary atresia.

Leptin signaling in neurotensin neurons involves STAT, MAP kinases ERK1/2, and p38 through c‐Fos and ATF1

The adipokine leptin signals energy status to the hypothalamus, which triggers a network of neuropeptide responses. Each hypothalamic cell type expresses a unique complement of neuropeptides, receptors, and second messengers; thus each likely responds specifically to peripheral hormones. We describe here the analysis of leptin signaling in a clonal population of mouse neurotensin (NT) -expressing hypothalamic neurons, N-39. Leptin induced phosphorylation of STAT3 and MAPK ERK1/2, but not the downstream effector of PI3K, Akt, and also induced c-Fos protein. We found activation of p38 MAPK by leptin, accompanied by phosphorylation of its downstream effector ATF-1. Phosphorylation of ATF-1 is blocked by the p38 MAPK inhibitor SB 203580. We linked this signaling directly to NT transcription. Protein binding analysis indicates that both ATF-1 and c-Fos are capable of binding to the mouse NT/N gene predominantly at physiological or high concentrations of leptin. The evidence indicates activation of distinct leptin signal transduction pathways that directly result in changes in NT gene expression and links these specific neurons to the control of energy homeostasis.

The effects of deleting the mouse neurotensin receptor NTR1 on central and peripheral responses to neurotensin.

Mice deficient in the neurotensin (NT)-1 receptor (NTR1) were developed to characterize the NT receptor subtypes that mediate various in vivo responses to NT. F2 generation (C57BL6/Sv129J) NTR1 knockout (-/-) mice were viable, and showed normal growth and overt behavior. The -/- mice lacked detectable NTR1 radioligand binding in brain, whereas NTR2 receptor binding density appeared normal compared with wild-type (+/+) mice. The gene deletion also resulted in the loss of NTR1 expression as determined by reverse transcription-polymerase chain reaction and in situ hybridization. Intracerebroventricular injection of NT (1 microg) to +/+ mice caused a robust hypothermic response (5-6 degrees C) and a significant increase in hot-plate latency. These effects were absent in the -/- mice. Similar results were obtained with i.p. injections of the brain-penetrant NT analog NMe-Arg-Lys-Pro-Trp-Tle-Leu (NT-2, 1 mg/kg i.p.). NT-2 administration also impaired rotarod performance in wild-type mice, but had no effect on motor coordination in knockout mice. In vitro, NT and NT-2 at 30 nM caused predominantly contraction and relaxation in isolated distal colon and proximal ileum, respectively, from +/+ mice, but no responses were observed with tissues from -/- mice. A similar loss of the contractile effects of NT was observed in the isolated stomach fundus from the knockout mice. In vivo, NT-2 administration reduced colonic propulsion substantially in wild-type mice. In contrast, NT-2 had no effect in NTR1 null mice, whereas the hypomotility effect of clonidine was intact. These data indicate that NTR1 mediates several of the central and peripheral effects of NT.

Mouse neurotensin receptor 2 gene ( Ntsr2): genomic organization, transcriptional regulation and genetic mapping on chromosome 12

We earlier isolated and characterized the mouse neurotensin receptor 1 ( Ntsr1) gene and developed Ntsr1 null mice. In the present study, we isolated the mouse neurotensin receptor 2 gene ( Ntsr2) and characterized the structure. The gene fragments available to us have 1.1 kb of 5′ upstream promoter region and a 7 kb coding region composed of four exons. Transcription initiation sites, determined by primer extension analysis, are located at 286 and 303 bp upstream from initiation of the ATG codon. The promoter region contains a TATA-like box, a typical CAAT box and putative GATA-2, CREB, Oct-2 and Ikarous 2 binding elements. We also found novel splice donor–acceptor sites for alternative splicing, which could generate a short form of mRNA encoding a truncate-type receptor. In addition, we determined the chromosomal location of the Ntsr2 gene and mapped it at 6 cM from the centromere on chromosome 12.

Pharmacological properties of the mouse neurotensin receptor 3. Maintenance of cell surface receptor during internalization of neurotensin

We recently reported the molecular identification of a new type of receptor for the neuropeptide neurotensin (NT), the neurotensin receptor 3 (NTR3), identical to sortilin, which binds receptor-associated protein. Here, we demonstrate that the cloned mouse NTR3 is expressed on the plasma membrane of transfected COS-7 cells. The mouse NTR3 is detectable by photoaffinity labeling and immunoblotting at the cell surface as a 100 kDa N-glycosylated protein. Biochemical analysis and confocal microscopic imaging clearly indicate that NT is efficiently internalized after binding to NTR3, and that despite this internalization, the amount of receptor present on the cell surface is maintained.

Contulakin-G, an O-Glycosylated Invertebrate Neurotensin

We have purified contulakin-G, a 16-amino acid O-linked glycopeptide (pGlu-Ser-Glu-Glu-Gly-Gly-Ser-Asn-Ala-Thr-Lys-Lys-Pro-Tyr-Ile-Leu-OH, pGlu is pyroglutamate) from Conus geographus venom. The major glycosylated form of contulakin-G was found to incorporate the disaccharide beta-D-Galp-(1-->3)-alpha-D-GalpNAc-(1-->) attached to Thr10. The C-terminal sequence of contulakin-G shows a high degree of similarity to the neurotensin family of peptides. Synthetic peptide replicates of Gal(beta-->3) GalNAc(alpha-->)Thr10 contulakin-G and its nonglycosylated analog were prepared using an Fmoc (9-fluorenylmethoxycarbonyl) protected solid phase synthesis strategy. The synthetic glycosylated con- tulakin-G, when administered intracerebroventricular into mice, was found to result in motor control-associated dysfunction observed for the native peptide. Contulakín-G was found to be active at 10-fold lower doses than the nonglycosylated Thr10 contulakin-G analog. The binding affinities of contulakin-G and the nonglycosylated Thr10 contulakin-G for a number of neurotensin receptor types including the human neurotensin type 1 receptor (hNTR1), the rat neurotensin type 1 and type 2 receptors, and the mouse neurotensin type 3 receptor were determined. The binding affinity of the nonglycosylated Thr10 contulakin-G was approximately an order of magnitude lower than that of neurotensin1-13 for all the receptor types tested. In contrast, the glycosylated form of contulakin-G exhibited significantly weaker binding affinity for all of the receptors tested. However, both contulakin-G and nonglycosylated Thr10 contulakin-G were found to be potent agonists of rat neurotensin receptor type 1. Based on these results, we conclude that O-linked glycosylation appears to be a highly unusual strategy for increasing the efficacy of toxins directed against neurotransmitter receptors.

Cross-tolerance between ethanol and neurotensin in mice selectively bred for ethanol sensitivity

Neurotensin (NT), a tridecapeptide that satisfies criteria as a neurotransmitter, mimics many actions of ethanol, and evidence indicates that some of the acute effects of ethanol are mediated in part by NT. Recent studies have shown that chronic ethanol treatment produced a downregulation of NT receptors in mesolimbic brain regions of long sleep (LS) mice and that reduced NT binding capacity was associated with acquisition and decay of tolerance to ethanol-induced locomotor inhibition and hypothermia in these mice. The present study was undertaken to determine whether cross-tolerance develops between NT and ethanol and whether chronic NT infusion produces NT receptor downregulation. Animals chronically treated with ethanol were tolerant to NT-mediated locomotor inhibition at a dose of 1.8 pmol NT, ICV, and were tolerant to NT-induced hypothermia at 1.8 and 6.0 pmol NT. Following repeated injections or continuous infusion of NT ICV, LS mice showed tolerance to both NT-and ethanol-induced hypothermia and locomotor inhibition. Indeed, ethanol doses that are hypnotic in control mice (2.8 g/ kg) were not effective in abolishing locomotor activity following chronic NT administration. Results with chronic saline infusion ICV indicate that stress alters sensitivity to ethanol-induced hypothermia. Chronic infusion of NT ICV produced a region-specific downregulation of high-affinity NT receptors in the striatum. The results demonstrate that cross-tolerance develops between NT and ethanol, and further support a role for neurotensinergic systems in the actions of ethanol.

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 stereoselectivity). 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.

Biochemical and pharmacological profile of a potent and selective nonpeptide antagonist of the neurotensin receptor.

We describe the characteristics of SR 48692, a selective, nonpeptide antagonist of the neurotensin receptor. In vitro, this compound competitively inhibits 125I-labeled neurotensin binding to the high-affinity binding site present in brain tissue from various species with IC50 values of 0.99 +/- 0.14 nM (guinea pig), 4.0 +/- 0.4 nM (rat mesencephalic cells), 7.6 +/- 0.6 nM (COS-7 cells transfected with the cloned high-affinity rat brain receptor), 13.7 +/- 0.3 nM (newborn mouse brain), 17.8 +/- 0.9 nM (newborn human brain), 8.7 +/- 0.7 nM (adult human brain), and 30.3 +/- 1.5 nM (HT-29 cells). It also displaces 125I-labeled neurotensin from the low-affinity levocabastine-sensitive binding sites but at higher concentrations (34.8 +/- 8.3 nM for adult mouse brain and 82.0 +/- 7.4 nM for adult rat brain). In guinea pig striatal slices, SR 48692 blocks K(+)-evoked release of [3H]dopamine stimulated by neurotensin with a potency (IC50 = 0.46 +/- 0.02 nM) that correlates with its binding affinity. In a cell line derived from a human colon carcinoma (HT-29), SR 48692 competitively antagonizes neurotensin-induced intracellular Ca2+ mobilization with a pA2 (-log Kapp) values of 8.13 +/- 0.03, which is consistent with results obtained in binding studies. Moreover, SR 48692 is devoid of any intrinsic agonist activity. This compound is also active in vivo, since it reverses at low dose (80 micrograms/kg) the turning behavior induced by intrastriatal injection of neurotensin in mice with similar potency whatever the route of administration (i.p. or orally) and with a long duration of action (6 hr). Thus, being a potent and selective neurotensin receptor antagonist, SR 48692 may be considered as a powerful tool for investigating the role of neurotensin in physiological and pathological processes.

Involvement of spinal monoaminergic pathways in antinociception produced by substance P and neurotensin in rodents

The antinociceptive effects of substance P and of neurotensin have been determined in rodents after depletion of serotonin (5-HT) or noradrenaline (NA) in the spinal cord. The antinociceptive effect of substance P, given intraventricularly, in rats and mice was blocked after depletion of 5-HT in the spinal cord with the neurotoxin 5,7-dihydroxytryptamine (5,7-DHT) or with the inhibitor of the synthesis of 5-HT, p-chlorophenylalanine (PCPA), but not after depletion of NA in the spinal cord with the neurotoxin 6-hydroxydopamine (6-OHDA). Conversely, the antinociceptive effect of neurotensin in mice was blocked after lesion of spinal NA pathways with 6-OHDA. When 5-HT spinal pathways of mice were lesioned with 5,7-DHT, neurotensin-induced antinociception was blocked 7 but not 15 days after the lesion. p-Chlorophenylalanine failed to prevent this effect of neurotensin. The results suggest that the antinociceptive effect of substance P depends on the integrity of spinal 5-HT neurones, whereas that of neurotensin depends on spinal NA neurones and, only to a limited extent, on 5-HT neurones. It seems that different descending systems are involved in the antinociception elicited by these two neuropeptides.

Adaptation to cold antagonizes neurotensin-induced hypothermia in mice

Intracerebrally-administered neurotensin produces a marked hypothermia in a variety of mammals. In this study, prior adaptation to a cold environment was found to significantly antagonize the hypothermia produced by intracisternally-administered neurotensin in mice. This antagonism required both previous exposure to cold ambient temperatures and cold exposure immediately prior to, or simultaneously with, neurotensin administration. The antagonism of neurotensin-induced hypothermia by prior cold-adaptation was blocked by indomethacin, but not by acetylsalicylic acid, suggesting that brain prostaglandin synthesis may be essential for this newly-discovered phenomenon.

Neurotensin: Antinocisponsive action in rodents

Neurotensin administered intracisternally in mice increased reaction time in the hot plate test (minimum effective dose = 25 ng) and decreased writhing induced by acetic acid (minimum effective dose = 0.25 ng). At the maximum effective dose of 250 ng, the antinocisponsive action of neurotensin lasted for about 1 h. In the hot plate test, neurotensin, [Gln 4]-neurotensin and [D-Ala 2]-methionine enkephalinamide were all equiactive at the three dose levels examined, 0.00015, 0.015 and 1.5 nmoles/mouse. At antinocisponsive doses, mice treated intracisternally with neurotensin also were hypothermic and had reduced locomotor activity. Neurotensin, 2500 ng, administered intravenously did not produce any of the effects described above. D,L-5-Hydroxytryptophan, oxotremorine and chlorpromazine given systemically caused hypothermia but did not change reaction time in the hot plate procedure. Neurotensin, even at doses as high as 2500 ng given intracisternally, did not disrupt coordinated motor control or change the current-response relationship for electroshock-induced seizures. The antinocisponsive action of neurotensin in mice (hot plate test) was not antagonized by naloxone, cyproheptadine, methergoline, atropine, benzotropine, mecamylamine, chlorpheniramine, pyribenzamine, cimetidine, HEAT, propranolol, phenoxybenzamine, haloperidol or indomethacin. Neurotensin administered intracisternally was also an effective antinocisponsive agent in rats, as assessed in the hot plate procedure, but not in the tail-flick test. Naloxone did not antagonize this action of neurotensin in the rat. It is suggested that the antinocisponsive effect of neurotensin results from an interaction of the peptide with neurotensin-specific receptor sites in the central nervous system.