Neurotensin Fragments Research Articles

Retrograde axonal transport of neurotensin in the dopaminergic nigrostriatal pathway in the rat

Although the existence of receptor transport has been clearly demonstrated in peripheral nerves, there is no clear cut evidence in the brain of such a process for neuropeptide receptors. Because of the localization of neurotensin receptors on dopaminergic terminals, the dopaminergic nigrostriatal pathway appears to be the system of choice for studying the axonal transport of neuropeptide receptors in the brain. When labelled neurotensin was injected into the rat striatum, a delayed accumulation of radioactivity in the ipsilateral substantia nigra was observed about 2 h after injection. An essential requirement to clearly observe this phenomenon was the pretreatment of animals with kelatorphan in order to prevent the labelled neurotensin degradation. The appearance of this labelling was prevented by injection of an excess of unlabelled neurotensin or of neurotensin 8–13, an active neurotensin fragment, but not by neurotensin 1–8, which had no affinity for neurotensin receptors. This process was saturable, microtubule-dependent and occurred only in mesostriatal and nigrostriatal dopaminergic neurons as identified after 6-hydroxydopamine lesion and by autoradiography.These results demonstrate that neurotensin was retrogradely transported by a process involving neurotensin receptors. The retrograde transport of receptor-bound neuropeptide may represent an important dynamic process which conveys information molecules from the synapse towards the cell body.

Neurotensin elevates cytosolic calcium in small cell lung cancer cells

The ability of neurotensin (NT) to elevate cytosolic Ca 2+ in small cell lung cancer (SCLC) cells was investigated using the fluorescent Ca 2+ indicator Fura 2-AM. Using SCLC cell line NCI-H345, NT elevated cytosolic Ca 2+ levels in a concentration-dependent manner. Using a 10 nM dose, NT and C-terminal fragments such as NT(8–13) but not N-terminal fragments such as NT(1–8) elevated the cytosolic Ca 2+ levels. Because EGTA (5 mM) did not affect the NT response, NT may cause release of Ca 2+ from intracellular stores. These data indicate that SCLC NT receptors may use Ca 2+ as a second messenger.

Biological potency of neurotensin metabolites in vivo: Importance of alcohol ‘fixation’ of blood

Neurotensin (NT), a 13-amino acid peptide, is released from the ileum following a meal. It is metabolized principally by the kidney and in the circulation to N-terminal fragments and apparently rapidly degraded C-terminal fragments. The present study was designed to compare the biological activity (plasma pancreatic polypeptide response) and the clearance kinetics of NT(1-13), the N-terminal fragment NT(1-11) and the C-terminal fragment NT(8-13). To measure accurately the circulating concentrations of short-lived NT fragments in the circulation, a method was devised of collecting blood directly into alcohol ('alcohol fixation'). The alcohol fixation procedure prevented the post-collection losses of the C-terminal fragment NT(8-13) and established that, based on blood levels achieved, NT(8-13) had 70% of the pancreatic polypeptide stimulating potency of NT(1-13). Nevertheless, the metabolic clearance rate of NT(8-13) was about sevenfold higher than the intact molecule, NT(1-13), suggesting that circulating C-terminal fragments have a minor physiological role. When the N-terminal fragment NT(1-11) was infused, there was no sustained effect on pancreatic polypeptide secretion although it was cleared at a rate similar to that of NT(1-13). It is concluded that the use of alcohol fixation prevents post-collection losses of NT fragments and enables true biological potencies of short-lived fragments to be assessed. The biological activity of NT resides in the C-terminus which, once split from the protective N-terminus, is rapidly degraded in the circulation. The remaining intact but inactive N-terminus is relatively stable.

Production and characterization of a novel monoclonal antibody against neurotensin: immunohistochemical localization in the midbrain and hypothalamus.

We developed a mouse monoclonal antibody against neurotensin (NT), termed NT8, for applications in immunohistochemistry and for ELISA analysis of NT. The antibody's paratope was determined by competitive ELISA using several peptide fragments of NT. That paratope requires intact peptide bonds between NT residues proline-7, arginine-8, and arginine-9. The antibody is of the IgG2B sub-isotype, having an IC50 for intact NT of approximately 3 nM when measured by competitive ELISA. Light microscopic immunohistochemical studies in the periaqueductal gray (PAG) and hypothalamus demonstrated staining patterns that agreed well with previous reports. Neuron perikarya were visualized even in the absence of colchicine pre-treatment, indicating that NT8 antibody is very sensitive in immunohistochemical applications. At the EM level, the antibody stained axon terminals, dendrites, and perikarya in the PAG. In lightly immunoreactive perikarya, rough endoplasmic reticula were visualized, suggesting that biosynthetic precursors to NT might be recognized by NT8.

Neutral endopeptidase modulates neurotensin-induced airway contraction.

To determine the role of endogenous neutral endopeptidase (NEP) (also called enkephalinase, EC 3.4.24.11) in regulating neurotensin-induced airway contraction, we used phosphoramidon, a specific NEP inhibitor, in the guinea pig. In studies in vitro, neurotensin and the COOH-terminal fragment neurotensin-(8-13) contracted strips of bronchial smooth muscle in a concentration-dependent fashion (P less than 0.001). In contrast, the NH2-terminal fragment neurotensin-(1-11) and the COOH-terminal fragment neurotensin-(12-13), the main fragments of neurotensin hydrolysis by NEP, had no effect. Phosphoramidon (10(-5) M) did not change resting tension but shifted the concentration-response curves to neurotensin to lower concentrations (P less than 0.001), whereas inhibitors of kininase II, aminopeptidases, serine proteases, and carboxypeptidase N were without effect. Removing the epithelium increased the contractile response to neurotensin (P less than 0.001), and phosphoramidon further increased the response to neurotensin in these tissues (P less than 0.001). Similar results were obtained in studies in vivo using aerosolized neurotensin and phosphoramidon. These results suggest that endogenous NEP in the airways modulates the effects of neurotensin on airway smooth muscle contraction by inactivating the peptide.

Neurotensin-induced polydipsia: a structure-activity study

Neurotensin (NT) and equimolar concentrations of NT fragments were microinjected into the lateral cerebral ventricle of water-deprived rats. NT (10μg) increased water intake 146% in the first 15 min following injection. While NT 1–11 and NT 10–13 had no effect on water intake, NT 8–13 increased water intake 101%. The polydipsia produced by NT 8–13 was statistically equivalent to that produced by NT. The results are discussed in relation to a proposed model of the NT receptor.

Neurotensin regulation of macrophage colony-stimulating factor-stimulated in vitro myelopoiesis.

Abstract Neurotensin, at less than or equal to 10(-9) M, in the presence of an optimal concentration of macrophage CSF (CSF-1), stimulated a dose-dependent enhancement of colony formation by murine marrow-derived mononuclear phagocyte progenitor cells. The additional colonies arose from the cell cycle and Ia Ag-positive subpopulation previously identified as two-signal-dependent progenitors. Two-signal colony formation diminished when the peptide was added at concentrations greater than 10(-9) M. Neurotensin binds specifically to two distinct receptors, a high affinity receptor (KD approximately 10(-9) M) and a lower affinity (KD approximately 10(-7) M) receptor identified as the tuftsin receptor. Rat liver ferritin and an inhibitory tuftsin analog. (ALA1)-tuftsin, which inhibit two-signal colony formation stimulated by tuftsin and tuftsin-like peptides in combination with CSF-1, did not inhibit colony formation stimulated by CSF-1 and 10(-9) M neurotensin. Both inhibitors, however, reversed the loss of two-signal colony growth in the presence of higher neurotensin concentrations. Neurotensin fragment 1-6, unlike ferritin and (ALA1)-tuftsin, inhibited two-signal colony formation stimulated by 10(-9) M neurotensin. However, like ferritin and (ALA1)-tuftsin, fragment 1-6 permitted full expression of two-signal colony formation in the presence of CSF-1 and 10(-7) M neurotensin. The data indicated that occupancy of both receptors at neurotensin concentrations greater than 10(-9) M might be responsible for the diminished progenitor response. The data further support a potential role for neurotensin as an inflammatory mediator. In addition to direct effects on mature phagocytic leukocytes, neurotensin, at least in vitro can influence the production of new mononuclear phagocytes.

The electrophysiological effects of neurotensin on neurones of guinea-pig prevertebral sympathetic ganglia.

1. The membrane effects of neurotensin on neurons of guinea-pig prevertebral ganglia were investigated by means of intracellular recording techniques in vitro. 2. Neurotensin (2-5 microM) applied by superfusion caused depolarizing responses in fifty-seven of seventy-four neurones tested in the inferior mesenteric ganglion and thirty-seven of forty-seven neurones tested in the coeliac plexus. The remaining neurones tested showed no membrane response. 3. Responses to neurotensin could be discriminated into two different types of membrane depolarizations on the basis of their different time courses and pharmacological characteristics: a steady-state type of depolarization and a transient type of depolarization. Seven of fifty-seven responsive neurones tested in the inferior mesenteric ganglion and ten of thirty-seven responsive neurones tested in the coeliac plexus responded to neurotensin with a depolarization which was maintained constant as long as neurotensin was superfused over the preparation (steady-state type). Forty-eight of fifty-seven responsive neurones tested in the inferior mesenteric ganglion and twenty of thirty-seven responsive neurones tested in the coeliac plexus responded with a transient depolarization which was followed by a repolarization in the maintained presence of neurotensin (transient type). A combination of both types of responses was observed in two neurones tested in the inferior mesenteric ganglion and in seven neurones tested in the coeliac plexus. 4. Steady-state type responses were characterized by a slowly developing membrane depolarization which reached a plateau and lasted throughout the presence of neurotensin. Amplitude and time course of this response were not altered in a solution containing hexamethonium (10 microM) and atropine (10 microM) or by a solution low in calcium (1 mM) and high in magnesium (15 mM). 5. Transient type depolarizations evoked by neurotensin were faster in reaching their maximum and were followed by a repolarization during the maintained presence of neurotensin. Responses similar in time course and amplitude were obtained in solutions containing hexamethonium (10-100 microM) and atropine (10 microM). However, transient responses were abolished in a solution low in calcium (1 mM) and high in magnesium (15 mM) and were markedly attenuated in ganglia treated with capsaicin (3 microM). 6. Both types of depolarizations were associated with increases in membrane input resistance. Both responses converted subthreshold depolarizing electrotonic potentials and subthreshold fast EPSPs to action potentials. 7. Both types of depolarizations were observed when the C-terminal hexapeptide fragment neurotensin 8-13 was used.(ABSTRACT TRUNCATED AT 400 WORDS)

The biological relevance of N-terminal neurotensin fragments in the regulation of exocrine pancreas secretion.

Stimulation of the exocrine pancreas is one of the biological effects attributed to the tridecapeptide neurotensin (NT). Only the C-terminal 8-13 fragment with its extremely short plasma half-life in the peripheral circulation was considered to be necessary for biological activity. The N-terminal fragments that are detectable in peripheral blood in much greater concentrations were considered to be of no importance. In order to elucidate the biological relevance of these N-terminal fragments, five dogs were infused with neurotensin at a rate (50 pmol/kg/h) that had been determined to give physiological plasma concentrations. Pancreatic secretions were collected from previously implanted modified Herrera pouches. The effects of the N-terminal fragments NT 1-8 and NT 1-11 on the exocrine pancreas secretion were tested in the same way. It was possible for the first time to demonstrate a biological effect of the N-terminal fragments found in the postprandial circulation in vivo. The plasma concentrations determined by radioimmunoassay remained within the physiological range (less than 40 pg/ml). These N-terminal neurotensin fragments may therefore participate in the hormonal regulation of the exocrine pancreas secretion.

Neurotensin receptors in the human spinal cord: A quantitative autoradiographic study

The anatomical localization of neurotensin receptors in the human spinal cord was examined in 12 cases aged 4–68 years using quantitative autoradiographic methods following the incubation of fresh, unfixed cryostat sections with 4 nM [ 3H]neurotensin. Characterization of the pharmacological specificity of the [ 3H]neurotensin binding sites in the human spinal cord from displacement studies with neurotensin 1–13 and various neurotensin fragments indicated that, whereas 1.0 μM neurotensin 1–13 and the car☐y-terminal fragment neurotensin 8–13 almost completely displaced [ 3H]neurotensin binding (4 nM), the ammo-terminal fragments neurotensin 1–8 and neurotensin 1–11 were weak inhibitors. This requirement for the car☐y-terminal fragment neurotensin 12–13 is consistent with [ 3H]neurotensin binding to specific neurotensin receptors in the human spinal cord. In all cases the autoradiograms demonstrated that neurotensin receptors were distributed in a similar fashion in the gray matter of the cervical, thoracic, lumbar, sacral and coccygeal regions of the human spinal cord. At all 21 spinal levels examined, the highest density of neurotensin receptors was localized in lamina II of the dorsal horn. Within lamina II the receptors were especially concentrated in the deeper inner segment (II j) where they formed a dense band lying immediately dorsal to lamina III. The density of receptors in this inner region of lamina II (23.5 fmol/mg) was almost double that in the outer segment of lamina II (12.2 fmol/mg), which showed the next highest density of receptors, and more than three times that in the adjacent lamina I (6.9 fmol/mg) and lamina III (7.1 fmol/mg). A moderate density of receptors was present in the intermediomedial (8.0 fmol/mg) and intermediolateral (8.0 fmol/mg) nuclei of lamina VII, and in lamina IX (4.4 fmol/mg). The density of labelling in the remaining laminae of the spinal cord was very low. These results indicate that neurotensin receptors are mainly localized in somatic and visceral sensory and motor regions of the human spinal cord and suggest that neurotensin may play a role in modulating sensory-motor functions in the human spinal cord.

Effects of ICV administration of neurotensin and analogs on EEG in rats.

The electroencephalographic (EEG) effects of the ICV administration of neurotensin (NT 1-13), NT 1-8 (an inactive neurotensin fragment) and D TYR-11 NT (a long-lasting analog of neurotensin) were studied in rats. In awake rats, NT 1-13 (30 micrograms) and D TYR-11 NT (10 micrograms) induced an increase of the power spectrum in the theta range activity (4-7 Hz). In rats recorded during the sleep-wakefulness cycles, NT 1-13 (10 and 30 micrograms) and D TYR-11 NT (10 micrograms) had an awakening effect and also induced an increase of latency to the first episode of the different sleep stages (intermediate stage and slow wave sleep). NT 1-8 (30 and 90 micrograms in awake rats, 10 and 90 micrograms for sleep-wakefulness cycles) was inactive in all these experiments. Thus, it seems that all these effects can be linked to neurotensin receptors; indeed only fragments which recognize receptors possess an EEG activity.

Peripheral inactivation of neurotensin. Isolation and characterization of a metallopeptidase from rat ileum.

A peptidase that inactivated neurotensin by cleaving the peptide at the Pro10-Tyr11 bond, generating the biologically inactive fragments neurotensin(1-10) and neurotensin(11-13) was purified from whole rat ileum homogenate. The purified enzyme behaved as a 70-75-kDa monomer as determined by SDS-PAGE analysis in reducing or non-reducing conditions and gel permeation on Ultrogel AcA34. The peptidase was insensitive to thiol-blocking agents and acidic and serine protease inhibitors but could be strongly inhibited by 1,10-phenanthroline, EDTA, dithiothreitol and heavy metal ions such as zinc, copper and cobalt. Zinc was the only divalent cation able potently to reactivate the apoenzyme. This enzyme could be distinguished from endopeptidases EC 3.4.24.15 and EC 3.4.24.11, angiotensin-converting enzyme, proline endopeptidase, aminopeptidase and pyroglutamyl-peptide hydrolase since it was not affected by micromolar concentrations of their specific inhibitors. The peptidase displayed a high affinity for neurotensin (1.6 microM). Studies concerning the specificity of the enzyme towards the sequence of neurotensin established the following. (a) Neurotensin(9-13) was the shortest partial sequence that fully inhibited tritiated neurotensin degradation; shortening the C-terminal part of the neurotensin molecule led to inactive fragments. (b) Amidation of the C-terminal end of the peptide did not prevent the recognition by the peptidase. (c) There existed a strong stereospecificity of the peptidase for the residues in positions 8, 9 and 11 of the neurotensin molecule. (d) Pro-Xaa dipeptides (where Xaa represented aromatic or hydrophobic residues) were the most potent inhibitors of tritiated neurotensin degradation while all the Xaa-Pro dipeptides tested were totally ineffective. (e) The neurotensin-related peptides: neuromedin N, xenopsin and [Lys8-Asn9]neurotensin(8-13), as well as angiotensins I and II and dynorphins(1-8) and (1-13) were as potent as neurotensin in inhibiting [3H]neurotensin hydrolysis.

Effect of neurotensin and its fragments neurotensin-(1–6) and neurotensin-(8–13) on dopamine release from cat striatum

At low concentrations, neurotensin (10 −9 M) enhanced electrically evoked release of dopamine. At higher concentrations, neurotensin (10 −7 M) also enhanced basal release of dopamine. The carboxy-terminal sequence of neurotensin-(8–13) was fully active and enhanced both electrically evoked and basal release. In contrast, the amino-terminal fragment neurotensin-(1–6) did not enhance electrically evoked release of dopamine even at high concentrations (10 −6 M). However, it retained the ability to enhance basal dopamine release. Combination of different doses of apomorphine with a subthreshold (10 −10 M) and submaximal concentrations (10 −9 M) of neurotensin gave clear additive effects. It is concluded that in the cat striatum neurotensin stimulates dopamine release by a direct effect on its own neurotensin receptor, and does not modulate the sensitivity of presynaptic dopamine autoreceptors. Furthermore, it is suggested that there exist at least two types of neurotensin receptors in the cat striatum. One type stimulates evoked dopamine release and another influences basal release of dopamine.

Pharmacological effects and metabolism of neurotensin and bradykinin in the isolated rat uterus

Neurotensin (NT) and bradykininin (BK) were found to cause contractions of isolated rat uterus preparations. In 97% of the experiments, acute tachyphylaxis followed soon after the initial administration of NT. Interrelation between the oxytocic effects of NT and BK was not observed. Among the NT fragments studied, only NT-(9–13) had an oxytocic effect (<1.0%). All NT fragments tested induced tachyphylaxis to NT regardless of their efficacy. Using HPLC analysis, NT but not BK was found to be degraded by the intact rat uterus. A major involvement of a carboxydipeptidase cleaving at Tyr 11-Ile 12 is suggested. Carboxyl-blocked neurotensinamide (NT-NH 2) was found to be resistent to proteolysis and not develop tachyphylaxis. No cross-tachyphylaxis was observed between NT and NT-NH 2. The oxytocic effects of NT-NH 2 were markedly potentiated by pre-incubation with NT or NT-(1–11). The results suggest the existence of different receptors for NT and BK in the uterus, as well as the existence of different receptors or receptor states that interact with NT or NT-NH 2 in the rat uterus.

The neurotensin analog xenopsin excites nigral dopamine neurons

The electrophysiological effects of neurotensin (NT), its analog xenopsin, and the fragment NT-(1-8) on the activity of dopaminergic neurons in rat substantia nigra slices were compared. Xenopsin and NT produced a concentration-dependent increase in the firing rate. The mean firing rate was increased by 58.5% with xenopsin (0.1 μM) and 49.1% with NT (0.1 μM). NT-(1-8) was inactive at this concentration. Dopamine (100 μM) inhibited firing in these neurons by 54.7%. These data provide evidence that xenopsin mimics the physiological effects of NT.

Further characterization of a neurotensin-degrading neutral metalloendopeptidase from rat brain

A peptidase inactivating neurotensin at the Pro 10-Tyr 11 peptidyl bond, leading to the biologically inactive fragments neurotensin 1–10 and neurotensin 11–13 was purified from rat brain homogenate. The peptidase was characterized as a 70 kDa monomer and could be classified as a metaliopeptidase with respect to its sensitivity to o- phenanthroline, EDTA and divalent cations. The enzyme was also strongly inhibited by dithiothreitol but appeared totally insensitive to thiol-blocking agents, acidic and serine protease inhibitors. Experiments performed with a series of highly specific peptidase inhibitors clearly indicated that the peptidase was a novel enzyme distinct from previously purified cerebral peptidases. The enzyme displayed a rather high affinity for neurotensin ( Km = 2.3 it M). Studies on its specificity indicated that: (i) neurotensin 9–13 was the shortest neurotensin fragment with full inhibitory potency of [ 3H]neurotensin degradation. Shortening the C-terminal end of the neurotensin molecule progressively led to inactive analogs; (ii) the peptidase exhibited a strong stereospecificity towards the residues in positions 8, 9 and 11. By contrast, neither introduction of a steric hindrance in position 11 nor amidation of the C-terminal end of the neurotensin molecule affected the ability of the corresponding analog to inhibit [ 3H]neurotensin degradation; (iii) Pro-Phe was the most potent dipeptide to compete for [ 3H]neurotensin degradation; (iv) the peptidase could not be described as an exclusive “neurotensinase” activity since, in addition to the neurotensin natural analogs (neuromedin N and xenopsin), non related natural peptides such as angiotensins I and II, dynorphins 1–8 and 1–13, atriopeptin III and bradykinin potently inhibited [ 3H]neurotensin degradation. Most of these peptides behaved as substrates for the enzyme.

Growth inhibition by neurotensin and neurotensin fragments in small cell lung cancer cell lines in vitro

Growth inhibition by neurotensin and neurotensin fragments in small cell lung cancer cell lines in vitro

High affinity binding of [ 3H]neurotensin to rat uterus

[ 3H]Neurotensin (NT) was found to bind specifically and with high affinity to crude membranes prepared from rat uterus. Scatchard analysis of saturation binding studies indicated that [ 3H]NT apparently binds to two sites (high affinity Kd 0.5 nM; low affinity Kd 9 nM) with the density of high affinity sites (41 fmoles/mg prot.) being about one-third that of the low affinity sites (100 fmoles/mg prot.). In competition studies, NT and various fragments inhibited [ 3H]NT binding with the following potencies (∼IC 50): NT 8–13 (0.4 nM), NT 1–13 (4 nM), NT 9–13 (130 nM), NT 1–11, NT 1–8 (>100 μM). Quantitatively similar results were obtained using brain tissue. These findings raise the possibility of a role for NT in uterine function.

Extraction of neurotensin-like immunoreactivities from porcine ileal mucosa

The extractability of neurotensin (NT) from porcine ileal mucosa was studied by comparison of eight extraction procedures. Tissue content of neurotensin-like immunoreactivity was quantitated and characterized by sequence-specific radioimmunoassays and gel filtration chromatography. Homogenization prior to boiling in extraction solvent produced higher levels of the intact peptide than the reverse procedure. N-terminal immunoreactivity was not influenced by the sequence of these steps. Tissue levels of intact NT were highest after extraction with 2.0 M acetic acid (mean 79.1 pmol/g, N=6 ) and lowest with distilled water (mean 6.5 pmol/g, N=6 ). The opposite was the case with levels of N-terminal immunoreactivity (mean 55.2 pmol/g and 105.7 pmol/g respectively, N=6 ). Recovery experiments with addition of synthetic NT 1–13 and the N-terminal fragment NT 1–8 indicated that these differences could be explained by differences in recovery of intact NT and N-terminal immunoreactive components in tissue. Gel chromatography confirmed that in acetic acid almost only the intact peptide was extracted from ileal mucosa, and showed that after extraction in water or phosphate buffer several N-terminal components were present. The results suggest that a molecular heterogeneity may be present in ileal tissue. If this concept is supported by further studies differential extraction procedures may be needed in the future.

Catabolism of neurotensin in the epithelial layer of porcine small intestine

The mammalian small intestine is both a source and a site of degradation of neurotensin. Metabolites produced by incubation of the peptide with dispersed enterocytes from porcine small intestine were isolated by high-performance liquid chromatography and identified by amino-acid analysis. The principal sites of cleavage were at the Tyr-11-Ile-12 bond, generating neurotensin-(1–11), and at the Pro-10-Tyr-11 bond, generating neurotensin-(1–10). The corresponding COOH-terminal fragments, neurotensin-(11–13) and -(12–13) were metabolized further. Formation of neurotensin-(1–11) and -(1–10) was completely inhibited by phosphoramidon ( K i = 6 nM), an inhibitor of endopeptidase 24.11, but not by captopril, an inhibitor of peptidyl dipeptidase A. Incubation of neurotensin with purified endopeptidase 24.11 from pig stomach also resulted in cleavage of the Tyr-11-Ile-12 and Pro-10-Tyr-11 bonds. A minor pathway of cell-surface-mediated degradation was the phosphoramidon-insensitive cleavage of the Tyr-3-Glu-4 bond, generating neurotensin-(1–3) and neurotensin-(4–13). No evidence for specific binding sites (putative receptors) for neurotensin was found either on the intact enterocyte or on vesicles prepared from the basolateral membranes of the cells. Neurotensin-(1–8), the major circulating metabolite, was not formed when neurotensin(1–13) was incubated with cells, but represented a major metabolite (together with neurotensin-(1–10) when neurotensin-(1–11) was used as substrate. The study has shown that degradation of neurotensin in the epithelial layer of the small intestine is mediated principally through the action of endopeptidase 24.11, but this enzyme is probably not responsible for the production of the neurotensin fragments detected in the circulation.