In vitro degradation of neurotensin in human plasma

To study the degradation of neurotensin in plasma in vitro, fresh human plasma was incubated with neurotensin in the presence and absence of the peptidase inhibitors pepstatin A, EDTA, PMSF and aprotinin. The half-time of disappearance of neurotensin at 37 degrees C was calculated to be 226 min in vitro as opposed to 1.4 min in vivo when measured by radioimmunoassay with a C-terminally directed neurotensin antiserum. Both gel filtration and reversed phase high-pressure liquid chromatography (HPLC) showed that the main degradation product of neurotensin in human plasma in vitro was chromatographically and immunologically identical to neurotensin 1-8 and HPLC also demonstrated the formation of neurotensin 1-11. The loss of neurotensin incubated in human plasma in vitro was greatly reduced by EDTA but not by the other peptidase inhibitors tested. In this respect peptidase(s) responsible for the degradation of neurotensin in plasma differ from those present in brain homogenates. EDTA may be of importance in the preservation of neurotensin in plasma samples.     

Plasma levels of neurotensin in gastroplasty for morbid obesity

Fasting and postprandial plasma levels of the tridecapeptide neurotensin were determined in ten women before and three months after gastroplasty for morbid obesity. Measurements were by radioimmunoassay in unextracted plasma with two antisera recognizing intact neurotensin (NT1-13) or intact neurotensin together with small C-terminal fragments, which may circulate as metabolites of neurotensin. Levels of both intact neurotensin and C-terminal immunoreactivity in obese women were in the same order of magnitude as those found previously in lean persons. Fasting levels measured with both antisera were significantly reduced following gastroplasty (P less than 0.01). Meal-stimulated levels and increments were unchanged. The cause of this prolonged reduction is at present unknown, but may be a reduced luminal stimulation of the small intestine or an altered vagal tonus following gastroplasty.     

Neurotensin inhibits neuronal Na+,K+-ATPase activity through high affinity peptide receptor

Neurotensin is a peptide present in mammalian CNS and peripheral tissues, which may play a major role in neurotransmission or neuromodulation, subserving diverse physiological functions. We studied the effect of added neurotensin on ATPase activities in synaptosomal membranes isolated from rat cerebral cortex. Neurotensin at 3 x 10(-8)-3 x 10(-6) M concentration decreased 20-44% Na+,K+-ATPase activity but failed to modify Mg2+-ATPase activity; lower neurotensin concentrations (3 x 10(-14)-3 x 10(-10) M) had no effect on enzyme activities. This inhibitory effect was abolished by neurotensin heating, by enzyme preincubation with neurotensin during periods exceeding 10 min, or by adding 1 x 10(-6) M SR 48692, a high affinity neurotensin receptor antagonist. Levocabastine, which blocks low affinity neurotensin receptor, failed to alter enzyme inhibition by the peptide. It is suggested that the sodium pump may be a target for neurotensin effects at neuronal level involving the participation of high affinity neurotensin receptor.     

Internalization and trafficking of neurotensin via NTS3 receptors in HT29 cells

The neurotensin receptor-3, originally identified as sortilin, is unique among neuropeptide receptors in that it is a single trans-membrane domain, type I receptor. To gain insight into the functionality of neurotensin receptor-3, we examined the neurotensin-induced intracellular trafficking of this receptor in the human carcinoma cell line HT29, which expresses both neurotensin receptor-1 and -3 sub-types. At steady state, neurotensin receptor-3 was found by sub-cellular fractionation and electron microscopic techniques to be predominantly associated with intracellular elements. A small proportion (approximately 10%) was associated with the plasma membrane, but a significant amount (approximately 25%) was observed inside the nucleus. Following stimulation with neurotensin (NT), neurotensin/neurotensin receptor-3 complexes were internalized via the endosomal pathway. This internalization entailed no detectable loss of cell surface receptors, suggesting compensation through either recycling or intracellular receptor recruitment mechanisms. Internalized ligand and receptors were both sorted to the pericentriolar recycling endosome/Trans-Golgi Network (TGN), indicating that internalized neurotensin is sorted to this compartment via neurotensin receptor-3. Furthermore, within the Trans-Golgi Network, neurotensin was bound to a lower molecular form of the receptor than at the cell surface or in early endosomes, suggesting that signaling and transport functions of neurotensin receptor-3 may be mediated through different molecular forms of the protein. In conclusion, the present work suggests that the neurotensin receptor-3 exists in two distinct forms in HT29 cells: a high molecular weight, membrane-associated form responsible for neurotensin endocytosis from the cell surface and a lower molecular weight, intracellular form responsible for the sorting of internalized neurotensin to the Trans-Golgi Network.     

The effect of oral ethanol ingestion on the diurnal neurotensin secretion in man.

Neurotensin is a tridecapeptide, present in the central nervous system and the gastrointestinal tract in man and animals. The affect of orally administered ethanol (1 g/kg body weight) on the neurotensin secretion over 24 hr period was studied in eight young healthy men. No significant circadian rhythm of neurotensin secretion was detected in the eight subjects studied. Ethanol produced a progressive rise in the plasma level of neurotensin reaching a maximum at 12:00 (13.8 +/- 8.6 pmol/l). At 12:00 and 14:00, the neurotensin concentrations were significantly higher than on the placebo day (p < 0.05). The secretion rate of neurotensin was determined approximately using the area under the curve method. Ethanol produced a transient rise in neurotensin secretion over the first 12 hrs period (08:00-20:00 h) after its administration (p < 0.02). The observation that ethanol increases transiently the neurotensin secretion in man supports the hypothesis that neurotensin may be involved in the biological effect of ethanol. The source of its secretion remains to be elucidated.     

Effects of mammalian and avian neurotensins and neurotensin fragments on wet-dog shaking and body temperature in the rat

The ability of mammalian and avian neurotensins and some neurotensin fragments to reduce wet-dog shaking (WDS) induced by thyrotrophin-releasing hormone (TRH) and to influence rectal temperature was tested after their injection into the periaqueductal grey region of male rats. Both neurotensins inhibited TRH-induced WDS and reduced rectal temperature by 2 degrees C; this latter effect was prevented by prior TRH administration. Of the four neurotensin fragments tested, both (1-8)- and (8-13)-neurotensin reduced WDS but only (8-13)-neurotensin reduced rectal temperature significantly. (1-6)- and (1-11)-neurotensin were without effect in either test system. From the activity of the various peptides, further examples of the mutual antagonism between TRH and neurotensin have been demonstrated. It is suggested that there is a possible role for neurotensin in controlling body temperature via the periaqueductal grey and that this may be one function of neurotensin in avian species; there may also be more than one receptor system binding neurotensin in the brain.     

大鼠纹状体边缘区内神经降压肽和生长抑素免疫阳性反应的分布

用免疫组织化学方法研究了大鼠纹状体边缘区中神经降压肽和生长抑素免疫阳性反应的分布。神经降压肽和生长抑素免疫阳性纤维和胞体散在分布于纹状体中,但在边缘区中分布密集,形成一条明显的带,带的宽度和位置和边缘区一致。边缘区中可见神经降压肽和生长抑素免疫阳性胞体。本研究证明纹状体边缘区中存在密度较高的神经降压肽和生长抑素免疫阳性纤维和胞体,并推测和边缘区的学习记忆功能有关     

Antinociceptive action of intrathecal neurotensin in mice

Neurotensin has been demonstrated to be analgesic in rodents. This study used intrathecal injection of neurotensin in unanesthetized mice to evaluate the effect of the peptide at the spinal level on unconditioned behavior. Intrathecal administration of neurotensin produced dose-related inhibition of locomotor activity and of the response elicited by subcutaneous hypertonic saline. The effects of the peptide in the tail flick assay were variable and it produced no inhibition of the behavioral response to intrathecal substance P. The results indicate that neurotensin antinociception at the spinal level does not result from locomotor impairment, may be somewhat selective for chemically induced pain, and may be mediated by a presynaptic action on primary afferent fibers.     

Isolation and characterization of a neurotensin-like decapeptide from a canine upper small intestinal extract

Neurotensin-like immunoreactivity can be detected in extracts of canine upper gastrointestinal mucosa when measured by carboxyl terminal but not by amino terminal antibodies to neurotensin. The nature of this immunoreactive material was characterized by complete purification on gel filtration and HPLC followed by peptide microsequence analysis. The structure obtained was Glu-Asn-Lys-Pro-Arg-Arg-Pro-Tyr-Ile-(Leu), identical in structure to the carboxyl terminal decapeptide of neurotensin. It cannot, however, be excluded that this neurotensin decapeptide was generated from a larger neurotensin-like peptide during the extraction procedure by a physiological or artificial enzymatic process. Since carboxyl terminal neurotensin fragments containing eight or more residues have full biological activity, this peptide may be responsible for neurotensin-like biological activities within the mucosa of, or after release from, the upper gut.     

Peripheral and Central Administration of Xenin and Neurotensin Suppress Food Intake in Rodents

Xenin is a 25‐amino acid peptide highly homologous to neurotensin. Xenin and neurotensin are reported to have similar biological effects. Both reduce food intake when administered centrally to fasted rats. We aimed to clarify and compare the effects of these peptides on food intake and behavior. We confirm that intracerebroventricular (ICV) administration of xenin or neurotensin reduces food intake in fasted rats, and demonstrate that both reduce food intake in satiated rats during the dark phase. Xenin reduced food intake more potently than neurotensin following ICV administration. ICV injection of either peptide in the dark phase increased resting behavior. Xenin and neurotensin stimulated the release of corticotrophin‐releasing hormone (CRH) from ex vivo hypothalamic explants, and administration of α‐helical CRH attenuated their effects on food intake. Intraperitoneal (IP) administration of xenin or neurotensin acutely reduced food intake in fasted mice and ad libitum fed mice in the dark phase. However, chronic continuous or twice daily peripheral administration of xenin or neurotensin to mice had no significant effect on daily food intake or body weight. These studies confirm that ICV xenin or neurotensin can acutely reduce food intake and demonstrate that peripheral administration of xenin and neurotensin also reduces food intake. This may be partly mediated by changes in hypothalamic CRH release. The lack of chronic effects on body weight observed in our experiments suggests that xenin and neurotensin are unlikely to be useful as obesity therapies.     

Revisiting the structure of the Vps10 domain of human sortilin and its interaction with neurotensin

Sortilin is a multifunctional receptor involved in sorting and apoptosis. We have previously reported a 2.0‐Å structure of the Vps10 ectodomain in complex with one of its ligands, the tridecapeptide neurotensin. Here we set out to further characterize the structural properties of sortilin and its interaction with neurotensin. To this end, we have determined a new 2.7 Å structure using a crystal grown with a 10‐fold increased concentration of neurotensin. Here a second peptide fragment was observed within the Vps10 β‐propeller, which may in principle either represent a second molecule of neurotensin or the N‐terminal part of the molecule bound at the previously identified binding site. However, in vitro binding experiments strongly favor the latter hypothesis. Neurotensin thus appears to bind with a 1:1 stoichiometry, and whereas the N‐terminus does not bind on its own, it enhances the affinity in context of full‐length neurotensin. We conclude that the N‐terminus of neurotensin probably functions as an affinity enhancer for binding to sortilin by engaging the second binding site. Crystal packing differs partly from the previous structure, which may be due to variations in the degree and pattern of glycosylations. Consequently, a notable hydrophobic loop, not modeled previously, could now be traced. A computational analysis suggests that this and a neighboring loop may insert into the membrane and thus restrain movement of the Vps10 domain. We have, furthermore, mapped all N‐linked glycosylations of CHO‐expressed human sortilin by mass spectrometry and find that their locations are compatible with membrane insertion of the hydrophobic loops.     

Molecular properties of neurotensin receptors in rat brain. Identification of subunits by covalent labeling

Neurotensin binding sites in rat brain synaptic membranes were specifically and covalently labeled by two methods. In the first, a photoreactive and highly radioactive analogue of neurotensin, 125I-labeled azidobenzoyl[Trp11]neurotensin, was synthesized and used to photoaffinity label neurotensin receptors. In the second, the reversible association between neurotensin receptors and 125I-labeled[Trp11]neurotensin, a radioactive but nonphotoreactive analogue of neurotensin, was made irreversible by means of disuccinimidyl suberate, a bifunctional cross-linking reagent. Analysis of synaptic membranes by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography revealed that using both methods the same two protein bands with apparent molecular weights of 49,000 and 51,000 were specifically labeled. Identical results were obtained with or without reduction of the photolabeled membranes by beta-mercaptoethanol before electrophoresis. Variation of the ligand concentration did not modify the relative labeling intensities of the two bands, indicating that the high- and low-affinity neurotensin binding sites previously detected in rat brain synaptic membranes have similar molecular structures. These results indicate that neurotensin receptors in rat brain may be composed of two different protein subunits with similar molecular weight of about 50,000, that are linked together by noncovalent bonds.     

Neurotensin modulates the composition of pancreatic exocrine secretions in chickens

The effects of neurotensin on pancreatic exocrine secretion were examined in fasted, conscious White Leghorn hens. A cannula was surgically implanted in the central duct serving the ventral lobe of the pancreas in order to collect pure pancreatic juice. Following recovery, neurotensin was infused intravenously at 3.6 or 10.8 pmol/kg*min. The volume and pH of the pancreatic secretions were recorded and total pancreatic protein concentration, amylase, lipase, trypsin, and chymotrypsin activity were measured every 30 min for 2 hr and compared to secretions following the infusion of 0.9% saline. Our results demonstrated that neurotensin did not affect the pH nor the pancreatic juice protein concentration, but did increase secretion rate following neurotensin infusion at 3.6 pmol/kg*min. Amylase activity was significantly depressed during neurotensin infusions, while lipase (both pancreatic and carboxylester lipase) activity was significantly elevated. The ratio of amylase to lipase activity was especially depressed by neurotensin infusion at 10.8 pmol/kg*min. Insufficient secretory activity prevented a balanced statistical analysis of chymotrypsin activity, but from a pooled analysis, neurotensin had no effect on protease activity in the pancreatic juice. These results support our current research indicating that neurotensin may be a hormonal regulator of postprandial lipid digestion in chickens.     

Stability of the Neurotensin Receptor NTS1 Free in Detergent Solution and Immobilized to Affinity Resin

Background

Purification of recombinant membrane receptors is commonly achieved by use of an affinity tag followed by an additional chromatography step if required. This second step may exploit specific receptor properties such as ligand binding. However, the effects of multiple purification steps on protein yield and integrity are often poorly documented. We have previously reported a robust two-step purification procedure for the recombinant rat neurotensin receptor NTS1 to give milligram quantities of functional receptor protein. First, histidine-tagged receptors are enriched by immobilized metal affinity chromatography using Ni-NTA resin. Second, remaining contaminants in the Ni-NTA column eluate are removed by use of a subsequent neurotensin column yielding pure NTS1. Whilst the neurotensin column eluate contained functional receptor protein, we observed in the neurotensin column flow-through misfolded NTS1.

Methods and Findings

To investigate the origin of the misfolded receptors, we estimated the amount of functional and misfolded NTS1 at each purification step by radio-ligand binding, densitometry of Coomassie stained SDS-gels, and protein content determination. First, we observed that correctly folded NTS1 suffers damage by exposure to detergent and various buffer compositions as seen by the loss of [³H]neurotensin binding over time. Second, exposure to the neurotensin affinity resin generated additional misfolded receptor protein.

Conclusion

Our data point towards two ways by which misfolded NTS1 may be generated: Damage by exposure to buffer components and by close contact of the receptor to the neurotensin affinity resin. Because NTS1 in detergent solution is stabilized by neurotensin, we speculate that the occurrence of aggregated receptor after contact with the neurotensin resin is the consequence of perturbations in the detergent belt surrounding the NTS1 transmembrane core. Both effects reduce the yield of functional receptor protein.     

Modulation by neurotensin and neuromedin N of adherence and chemotaxis capacity of murine lymphocytes.

The action of neurotensin and related peptides has not been yet studied on lymphocytes, although there are studies indicating the stimulative action of neurotensin, a peptide first isolated from bovine hypothalamus, on different functions of phagocytic immune cells. The present study demonstrates that neurotensin and a related peptide, neuromedin N, increased significantly the adherence and chemotaxis capacity of murine peritoneal lymphocytes, when they were incubated in the presence of neuropeptide concentrations between 10(-9) M and 10(-12) M. With respect to their adherence capacity, neuromedin N showed a slightly higher stimulation than neurotensin at a shorter time. However, both neuropeptides stimulated the chemotaxis capacity in a similar percentage. The study of the action mechanisms of these neuropeptides showed that intracellular cAMP levels were not modified by neurotensin or neuromedin N, but using an extracellular calcium chelator, EGTA (1 mM), and a blocker of calcium channels in endoplasmic reticulum, ryanodine (0.5 mM), we observed that neurotensin and neuromedin N could produce their effects through an augmentation of the intracellular Ca2+ concentration. As adherence and chemotaxis are initial processes of immune response, the results show that both neuropeptides may be physiological modulators of the lymphocyte function.     

Marsupial possum neurotensin: a unique mammalian regulatory peptide exhibiting structural homology to the avian analogue

Neurotensin has been isolated from small intestinal extracts of an Australian marsupial, the brush-tailed possum (Trichosurus vulpecula). The primary structure was determined as: pGlu-Leu-His-Val-Asn-Lys-Ala-Arg-Arg-Val-Tyr-Ile-Leu. When compared with bovine neurotensin, marsupial possum neurotensin exhibits four amino acid substitutions. His for Tyr3, Val for Glu4 and Ala for Pro7 are identical with those found in chicken neurotensin. In addition, substitution of Pro10 with Val is unique among all neurotensins sequenced to date. Marsupial possum neurotensin is therefore of unique primary structure, displaying most sequence homology with its avian counterpart. This neurotensin may thus resemble the phylogenetic precursor present at the time of divergence of primitive mammals and birds.     

Neurotensin interacts with carbachol, secretin, and caerulein in the stimulation of the exocrine pancreas of the rat in vitro

In the present investigation the effect of neurotensin on pancreatic secretion of isolated pancreatic lobules from the rat was examined. We found a dose- and time-dependent stimulation of amylase release beginning with a concentration of 10(-9) M neurotensin. This response was potentiated by the cholinergic agonist carbachol, the gastrointestinal peptide secretin, and the CCK analogue caerulein. As we found neurotensin-immunoreactive nerves within the pancreas and as neurotensin-like immunoreactivity is present in the circulation (found previously), neurotensin may well be a further peptide taking part in the regulation of exocrine pancreatic secretion either as a hormone or a neurotransmitter. Neurotensin would then cooperate with cholinergic mechanisms, secretin, and CCK.     

Receptors for neurotensin in canine small intestine.

We have previously characterized the neurotensin receptors on the circular smooth muscle (CM) of the canine small intestine (1). In the present studies, using radioligand binding technique, neurotensin receptors were localized on the membranes from deep muscular (DMP) and the submucous plexus while no binding was observed on either the longitudinal smooth muscle or myenteric plexus membranes. The high affinity binding sites (Kd 0.1-0.2 nM) on DMP membranes were similar to those on CM; the low affinity component was of much lower affinity (Kd approximately 40 nM). DMP had 4-6 times higher density of binding sites than the CM. The recognition properties of DMP receptors were similar to those on the CM and reduced sulfhydryl groups were required for the binding activity. The action of neurotensin on the contractility of the canine small intestine, therefore, appears to be through a direct action on the circular smooth muscle and through the prejunctional action on the DMP neurons through distinct receptors. Thiol groups in the neurotensin receptors may be important for the receptor function.     

Psychostimulant-like effect of central microinjection of neurotensin on brain stimulation reward

The curve shift method and the brain stimulation reward paradigm were used to dissociate reward and performance changes and determine whether unilateral ICV microinjection of neurotensin (3, 10, and 30 μg/10 μl) produces neuroleptic- or psychostimulant-like effect on a dopamine-dependent behavior. At the highest dose tested, neurotensin potentiated brain stimulation reward, producing a significant time-dependent decrease in frequency threshold. Neurotensin also suppressed maximal rate of responding at every dose tested, suggesting that it was more effective at attenuating performance capability. These results suggest that a centrally acting neurotensin receptor agonist may specifically stimulate dopamine-dependent behaviors, producing psychostimulant-like effect that can be attenuated or masked by a suppression of performance capability.     

On the effect of xenin and xenin fragments on exocrine pancreas secretion in vivo.

A stimulatory effect on exocrine pancreas secretion could be demonstrated with high concentrations of the 25-amino-acid peptide xenin in non-anesthetized dogs. This peptide has been isolated from gastric mucosa and it is part of a structural coat protein. It has close structural similarities to neurotensin. The longer C-terminal fragments xenin-(13--25) and xenin-(18--25) are essential for the stimulation of exocrine pancreas secretion in vivo. The smaller peptide fragments xenin-(21--25) and xenin-(22--25) failed to stimulate the pancreas as well as the N-terminal peptide fragment xenin-(1--23). The stimulatory effects of xenin may be mediated via neural neurotensin pathways, because neurotensin receptor blockade abolished the stimulatory effect on pancreatic secretion. Cholinergic pathways are not involved, because atropine had no inhibiting effect.