Isolation and primary structure of an amphibian neurotensin.

Using a radioimmunoassay system employing an antiserum which recognises the common C-terminal tripeptide (YIL) of neurotensin (NT) and neuromedin N (NN), immunoreactivity was identified in extracts of brain (65.8 pmol/g), small intestine (44.2 pmol/g) and rectum (13.2 pmol/g) of the European common frog (Rana temporaria). No immunoreactivity was detected in extracts of stomach and skin. Reverse-phase HPLC analysis of each tissue extract resolved a single immunoreactive peptide with identical retention time in each case. The immunoreactive peptide was isolated by reverse-phase HPLC from brain extracts and an N-terminal pyroglutamyl residue was successfully removed enzymatically. The molecular mass of des(pyroglutamyl) frog NT, determined by plasma desorption mass spectroscopy, was 1440 Da. The primary structure of this peptide was determined by gas-phase sequencing and the calculated molecular mass, 1440.7 Da, was in close agreement with that derived by mass spectroscopy. The full primary structure of frog NT was established as: QSHISKARRPYIL. When compared with bovine NT, frog NT exhibits five amino acid substitutions in the N-terminal region, whereas the C-terminal hexapeptide sequence (RRPYIL), which mediates the classical biological effects of NT, is completely conserved. Amphibia thus possess a tridecapeptide NT which is analogous to that of higher vertebrates and considerable constraints on the primary structure of the C-terminal biologically-active core have existed for a vast evolutionary time span.     

Proneurotensin 1-117, a stable neurotensin precursor fragment identified in human circulation

Proneurotensin/neuromedin N (pro NT/NMN) is the common precursor of two biologically active peptides, neurotensin (NT) and neuromedin N (NMN). We have established antibodies against peptide sequences of the NT/NMN precursor and developed a sandwich immunoassay for the detection of pro NT/NMN immunoreactivity in human circulation. Endogenous pro NT/NMN immunoreactivity was enriched by affinity chromatography using antibodies against two different pro NT/NMN epitopes, and further purified by reversed phase HPLC. Mass spectrometry analysis revealed pro NT/NMN 1-117 as major pro NT/NMN immunoreactivity in human circulation. Pro NT/NMN 1-117 is detectable in serum from healthy individuals (n = 124; median 338.9 pmol/L). As known for NT, the release of pro NT/NMN 1-117 from the intestine into the circulation is stimulated by ingestion of an ordinary meal. Investigation of the pro NT/NMN 1-117 in vitro stability in human serum and plasma revealed that this molecule is stable for at least 48 h at room temperature. Since pro NT/NMN 1-117 is theoretically produced during precursor processing in stoichiometric amounts relative to NT and NMN, it could be a surrogate marker for the release of these bioactive peptides.     

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.     

Isolation and quantitation of several new peptides from the canine neurotensin/neuromedin N precursor

Using antisera towards the bioactive peptides, neurotensin and neuromedin N, as well as towards the N-terminal and C-terminal regions of their shared 170-residue precursor, peptides representing various portions of the precursor were isolated from extracts of canine ileum. In total, seven peptides were isolated, two of which had not been previously identified. One was the C-terminal tail of the precursor (Gly-Ser-Tyr-Tyr-Tyr) and the other was the tail peptide extended N-terminally to include neurotensin (Glp-Leu-Tyr-Glu-Asn-Lys-Pro-Arg-Arg-Pro-Tyr-Ile-Leu-Lys-Arg-Gly-Ser-Tyr-Tyr-Tyr). By comparing the measured concentrations for each of the identified peptides, it was established that processing at the three Lys-Arg cleavage sites within the precursor did not occur to the same extent. Cleavage at the N-terminus of neuromedin N was 10% complete, that between neurotensin and the tail was 90% complete, and that between neuromedin N and neurotensin was 95% complete. Three immunoreactive proteins were also identified by immunochemical and chromatographic analyses: N-terminally extended neuromedin N (125 residues), N-terminally extended neurotensin (140 residues), and the entire 147-residue precursor. It was concluded that neurotensin, tail and large molecular neuromedin N were the primary products of processing for this precursor in canine ileum, while minor products included neuromedin N, neurotensin tail, and large molecular neurotensin.     

Production of recombinant large proneurotensin/neuromedin N-derived peptides and characterization of their binding and biological activity.

Proneurotensin/neuromedin N (pro-NT/NN) is the common precursor of two biologically active related peptides, neuromedin N (NN) and neurotensin (NT). It undergoes a tissue-specific processing leading to the formation in some tissues and cancer cell lines of large peptides ending with the NT (large NT) or NN (large NN) sequence. In this study, we prepared and purified high amounts of recombinant large NT and large NN using the Drosophila S2 cell expression system. The binding and pharmacological properties of recombinant large peptides were characterized and compared to those of NT and NN using either COS cells transfected with the human subtype-1 NT receptor (hNTS1) or the human colon adenocarcinoma HT29 cell line that endogenously expresses hNTS1. Furthermore, the metabolic stability of the large peptides, when exposed to HT29 cells, was compared to that of NT and NN. Both large NT and large NN were able to bind to and activate hNTS1 with potencies that were approximately 10 times lower than that of their small counterpart. In addition, the large forms proved to be far less sensitive to degradation than the small peptides. Taken together, these data suggest that the large forms might represent endogenous, long-lasting activators of hNTS1 in a number of physiopathological situations.     

Compared Binding Properties of 125I-Labeled Analogues of Neurotensin and Neuromedin N in Rat and Mouse Brain

Abstract: Neurotensin and neuromedin N are two structurally related peptides that are synthesized by a common precursor. The purpose of the present work was to characterize neuromedin N receptors in rat and mouse brain and to compare these receptors with those of neurotensin. A radiolabeled analogue of neuromedin N has been prepared by acylation of the N-terminal amino group of the peptide with the ¹²⁵I-labeled Bolton-Hunter reagent. This ¹²⁵I-labeled derivative of neuromedin N bound to newborn mouse brain homogenate with high affinity (K _d = 0.5 n M ). Cross-competition experiments between radiolabeled and unlabeled neurotensin and neuromedin N indicated that each peptide was able to displace completely and specifically the other peptide from its interaction with its receptor. Independently of the radioligand used, the affinity of neurotensin was always better than that of neuromedin N. Quantitative radioautographic studies demonstrated that the ratio of labeling intensities obtained with ¹²⁵I-labeled analogues of neurotensin and neuromedin N remained constant in all the brain areas. Our results do not support the existence of a specific neuromedin N receptor in rat and mouse brain and can be explained by the presence of a common receptor for both peptides.     

Neurotensin-like immunoreactivities in human plasma: feeding responses and metabolism

Feeding responses and day and night levels of plasma concentration of neurotensin (NT) and NT-fragments were studied in healthy subjects. Plasma levels were measured by three radioimmunoassays recognizing intact NT in addition to C- and N-terminal immunoreactivity. The metabolism of NT was studied following intravenous administration. In 106 subjects fasting levels of intact NT (median 18 pmol/l), C-terminal (median 30 pmol/l) and N-terminal immunoreactivity (median 95 pmol/l) were unrelated to sex or age. Postprandially plasma levels in seven subjects measured with all assays increased by a factor 1-3. Following a mixed meal the increase was biphasic, whereas the response to dairy cream was monophasic. Repetitive measurements during 24 hours showed that levels of N-terminal immunoreactivity fluctuated in a manner related to meal ingestion and were elevated throughout the daytime, whereas intact NT and C-terminal immunoreactivity changed little. Following intravenous infusion of 2.4 pmol/kg/min NT in 5 subjects the chromatographic pattern was similar to that seen postprandially. The plasma half life of intact NT and C-terminal immunoreactivity was 1.5 and 1.2 min, whereas that of N-terminal immunoreactivity was 10.0 min. The differences in circulating levels could be explained by these differences in metabolism, but the physiological significance remains to be elucidated.     

Enhanced expression of neurotensin/neuromedin N mRNA and products of NT/NMN precursor processing in neonatal rats

Intestinal levels of immunoreactive neurotensin (iNT) and neuromedin N (iNMN), as well as mRNA for the NT/NMN precursor, were elevated during the suckling period in rats. While transient expression of NT/NMN was observed at 1–5 days of age in the proximal small intestine and colon, NT/NMN levels in the ileum increased to peak at 10–20 days of age and then decreased to adult levels. The levels of these peptides were not elevated in the central nervous system and pituitary over this time period. Chromatographic analyses of jejunoileal extracts indicated that large molecular forms of iNT and iNMN were present, constituting 1.3% of total iNT and 56% of total iNMN, respectively. Treatment of the large forms with pepsin, which is known to generate the fully immunoreactive peptides, NT(3–13), NT(4–13), and NMN, increased immunoreactivity tenfold (iNT) and 1.2-fold (iNMN). Thus, large forms actually constituted 13% (iNT) and 60% (iNMN). Based upon its physicochemical properties, large molecular iNMN was tentatively identified as a 125 residue peptide with NMN at its C-terminus [i.e., rat prepro-NT/NMN(23–147)]. The properties of large molecular iNT were most similar to those predicted for the entire precursor [i.e., rat prepro-NT/NMN(23–169)]. These results indicate a) that enhanced expression of NT/NMN occurs in a tissue-specific manner in rats during the suckling period; b) that the pattern of precursor processing in intestine yields primarily NT and a large molecular form of NMN.     

Rabbit neuromedin U-25: lack of conservation of a posttranslational processing site

The rabbit small intestine contains neuromedin U-like immunoreactivity (22 pmol/g wet tissue weight) that was resolved into a single major molecular form by reversed-phase HPLC. The primary structure of the peptide was established as: Phe-Pro-Val-Asp-Glu-Glu-Phe-Gln-Ser-Pro10-Phe-Gly-Ser-Arg-Ser-Arg- Gly-Tyr-Phe- Leu20-Phe-Arg-Pro-Arg-Asn.NH2. In rabbit neuromedin U, the Arg16-Arg17 dibasic residue processing site that is found in pig and dog neuromedin U-25 is replaced by Arg-Gly, but this potential monobasic processing site is not utilized by cleavage enzyme(s) in the intestine.     

Neuromedin N: presence and chromatographic characterization in the rat

The distribution of neuromedin N and its structurally related peptide, neurotensin, was investigated in the rat and found to be remarkably similar with highest concentrations in the ileum. However, neuromedin N but not neurotensin was found in the kidney. Chromatographic analysis of immunoreactive neuromedin N demonstrated a single peak of immunoreactivity which was distinguishable from the single peak of immunoreactive neurotensin. Neuromedin N is likely to be a naturally occurring peptide and is distinct from neurotensin in rat peripheral tissues.     

Differential processing of pro-neurotensin/neuromedin N and relationship to pro-hormone convertases

Neurotensin (NT) is synthesized as part of a larger precursor that also contains neuromedin N (NN), a six amino acid neurotensin-like peptide. NT and NN are located in the C-terminal region of the precursor (pro-NT/NN) where they are flanked and separated by three Lys-Arg sequences. A fourth dibasic sequence is present in the middle of the precursor. Dibasics are the consensus sites recognized and cleaved by endoproteases that belong to the recently identified family of pro-protein convertases (PCs). In tissues that express pro-NT/NN, the three C-terminal Lys-Arg sites are differentially processed, whereas the middle dibasic is poorly cleaved. Pro-NT/NN processing gives rise mainly to NT and NN in the brain, to NT and a large peptide ending with the NN sequence at its C-terminus (large NN) in the gut and to NT, large NN and a large peptide ending with the NT sequence (large NT) in the adrenals. Recent evidence indicates that PC1, PC2 and PC5-A are the pro-hormone convertases responsible for the processing patterns observed in the gut, brain and adrenals, respectively. As NT, NN, large NT and large NN are all endowed with biological activity, the evidence reviewed here supports the idea that post-translational processing of pro-NT/NN in tissues may generate biological diversity.     

Differential processing of neurotensin/neuromedin N precursor(s) in canine brain and intestine

By using a radioimmunoassay for neuromedin N (NMN), a hexapeptide in the neurotensin (NT) family, extracts of canine small intestine were found to contain primarily (greater than 75%) large molecular form(s) of NMN, whereas the predominant species in brain was NMN itself. Large NMN was highly basic (pI greater than 9) and during sodium dodecyl sulfate gel electrophoresis gave two components of approximately 17 kDa (75%) and approximately 8 kDa (25%). Large NMN, like NT, was localized primarily to the mucosal layer of the jejunoileum. It was also present in highly purified (25% pure) mucosal N-cells, where it appeared to be concentrated within dense secretory vesicles. The amino acid sequence of a 21-amino acid fragment cleaved from the C-terminal region of large NMN was identical to residues 128-148 of the canine NT/NMN precursor predicted from cDNA work. These results suggest that tissue-specific processing of the NT/NMN precursor occurs in the dog, giving rise to NMN in brain and large NMN in small intestine.     

Different relative abundance of neurotensin and neuromedin N in bovine ocular tissues.

Neurotensin (NT) and neuromedin N (NN) are regulatory peptides encoded by the same gene and located in tandem within a common precursor. Using specific radioimmunoassays for both peptides, their relative abundance in extracts of bovine ocular tissues has been examined. Within the retina, the molar concentration of NN was significantly higher (P less than 0.001) than that of NT. In contrast, within both choroid/sclera and iris/ciliary bodies, the molar concentration of NT was significantly higher (P less than 0.001) than that of NN. These data demonstrate that the theoretical molar ratio of 1:1, which would result from complete processing of both peptides from the common precursor, does not occur in any of the ocular tissues examined. Reverse phase HPLC of extracts of each ocular tissue confirmed the differential abundance of NT and NN. These data would suggest that the common NT/NN precursor is differentially-processed within bovine ocular tissues, a finding which may be of physiological significance.     

Isolation and microsequence analysis of a novel form of neuromedin U from guinea pig small intestine

A multidimensional chromatographic regimen has been used to isolate and purify a peptide showing immunoreactivity for neuromedin U from guinea pig small intestine. Microsequence Edman N-terminal analysis and C-terminal analysis by enzymatic digestion showed this peptide to be a nonapeptide with the following sequence: H-Gly-Tyr-Phe-Leu-Phe-Arg-Pro-Arg-Asn-NH2. The C-terminal octapeptide of this sequence is the same as porcine NMU-8, and the C-terminal heptapeptide is identical to rat NMU(17-23).     

The role of dibasic residues in prohormone sorting to the regulated secretory pathway. A study with proneurotensin

The mechanisms by which prohormone precursors are sorted to the regulated secretory pathway in neuroendocrine cells remain poorly understood. Here, we investigated the presence of sorting signal(s) in proneurotensin/neuromedin N. The precursor sequence starts with a long N-terminal domain followed by a Lys-Arg-(neuromedin N)-Lys-Arg-(neurotensin)-Lys-Arg- sequence and a short C-terminal tail. An additional Arg-Arg dibasic is contained within the neurotensin sequence. Mutated precursors were expressed in endocrine insulinoma cells and analyzed for their regulated secretion. Deletion mutants revealed that the N-terminal domain and the Lys-Arg-(C-terminal tail) sequence were not critical for precursor sorting to secretory granules. In contrast, the Lys-Arg-(neuromedin N)-Lys-Arg-(neurotensin) sequence contained essential sorting information. Point mutation of all three dibasic sites within this sequence abolished regulated secretion. However, keeping intact any one of the three dibasic sequences was sufficient to maintain regulated secretion. Finally, fusing the dibasic-containing C-terminal domain of the precursor to the C terminus of beta-lactamase, a bacterial enzyme that is constitutively secreted when expressed in neuroendocrine cells, resulted in efficient sorting of the fusion protein to secretory granules in insulinoma cells. We conclude that dibasic motifs within the neuropeptide domain of proneurotensin/neuromedin N constitute a necessary and sufficient signal for sorting proteins to the regulated secretory pathway.     

Release of ileal neurotensin in the rat by neurotransmitters and neuropeptides.

To investigate the functional relationship between the enteric nervous system and the intestinal neurotensin (N) cells, the release of neurotensin (NT) was measured upon vascular 8-min infusion periods of various neurotransmitters and neuropeptides in an isolated vascularly perfused rat jejunoileum. NT-like immunoreactivity (NT-LI) was measured with an antiserum that specifically recognizes intact NT. The cholinergic agonists methacholine and carbachol produced a strong release of NT-LI (250% and 700% of basal, respectively at 10(-5) M). The infusion of a lower dose (10(-7) M) was less effective in both cases. The nicotinic receptor agonist DMPP (10(-4) M) had no significant effect on NT-LI release. Norepinephrine (10(-6) M) produced a moderate and well-sustained secretion of NT (200% of basal). Infusion of higher doses of these neurotransmitters dramatically increased the arterial pressure. G-amino-n-butyric acid (GABA), histamine, serotonin and dopamine administered at final concentrations up to 10(-5) M had no effect on NT-LI release. In contrast, gastrin-releasing peptide and bombesin induced a dose-dependent transient increase of portal NT-LI (maximal value at 10(-7) M: 1000% of basal) followed by a rapid return to near basal values. Substance P (10(-7) M) evoked a prompt release of NT-LI with a peak at 600% of basal followed by a decline to 200% of basal at the end of the session. Leu-enkephalin and calcitonin-gene-related-peptide (CGRP, 10(-7) M) produced a small rise in portal NT-LI, while Met-enkephalin, dynorphin, vasoactive intestinal peptide (VIP), galanin, neuropeptide Y (NPY), peptide histidine isoleucine (PHI), neuromedin U and thyrotropin releasing hormone (TRH) had no stimulatory effect. Our results indicate that additionally to the secretion of NT induced by cholinergic agents and bombesin, substance P and to a lesser extent Leu-enkephalin are capable of stimulating NT release in the rat.     

Canine neurotensin, neurotensin6-13 and neuromedin N: primary structures and receptor activity

Canine neurotensin (NT) and neuromedin N (NMN) were isolated from extracts of ileal mucosa using radioimmunoassay for detection. The structures determined were consistent with those predicted by earlier cDNA work. The molar ratio of NT to NMN was ca. 7, suggesting that the NT/NMN precursor, which contains one copy of each peptide, undergoes complex posttranslational processing or that other NT-precursors lacking NMN exist. In addition to NT, small quantities of NT6-13 and NT2-13 were obtained. Native and synthetic preparations of these peptides were indistinguishable in a radioreceptor assay employing rat brain membranes and 125I-labeled NT; NT6-13 was ca. 8-times more potent than NT and NMN was about one-sixth as potent as NT. NT6-13 was also ca. 10 times more potent than NT in inhibiting spontaneous contractile activity in longitudinally-oriented smooth muscle strips of porcine jejunum. Preparations of intestinal N-cells as well as N-cell vesicles also appeared to contain NT2-13 and NT6-13; however, it is not yet clear whether these peptides are utilized physiologically or simply represent metabolites of NT. These results suggest that further work on the processing of NT precursor and on biologic abilities of partial sequences of NT could be fruitful.     

Neuromedin U--a study of its distribution in the rat

The distribution of neuromedin U, a novel peptide originally isolated from porcine spinal cord, was investigated in the rat using a recently developed radioimmunoassay. High concentrations of neuromedin U-like immunoreactivity were found in the pituitary gland and gastrointestinal tract. Significant concentrations of immunoreactivity were also found in several regions of the rat brain, spinal cord and both male and female genitourinary tracts. In the small intestine, neuromedin U-like immunoreactivity was restricted to the submucosal muscular layers, suggesting localization in neurones rather than in epithelial cells. Chromatographic analysis of pituitary, spinal cord and gut revealed a single peak of immunoreactivity which did not co-elute with either synthetic porcine neuromedin U-25 nor neuromedin U-8, indicating inter-species molecular heterogeneity.     

Potentiation by thiorphan and bestatin of the naloxone-insensitive analgesic effects of neurotensin and neuromedin n

Neurotensin induced significant antinociceptive activity as measured in a variety of nociceptive tests 10 and 30 min following intracerebroventricular (i.c.v.) injection in mice. The lowest effective peptide doses were 25 ng in the writhing test, 25–50 ng in the tail-flick test, 50–100 ng in the hot-plate test and 2000 ng in the tail electrical stimulation test. The neurotensin related hexapeptide neuromedin N also displayed antinociceptive properties but only in the writhing and tail-flick tests. Furthermore, as compared to neurotensin, the neuromedin effects required higher doses. ED₅₀'s for neurotensin and neuromedin in the writhing test were 70 ng and 1070 ng, respectively. Separate or combined injections of the endopeptidase 24.11 (enkephalinase) inhibitor thiorphan (l0μg) and the aminopeptidase inhibitor bestatin (50μg) did not affect tail-flick latencies. In contrast, i.c.v. injection of thiorphan together with an ineffective dose of neurotensin (25 ng) resulted in a significant antinociceptive effect. Bestatin did not modify tail-flick latencies in neurotensin-treated mice whether in the absence or presence of thiorphan. On the contrary, each of these peptidase inhibitors promoted antinociceptive effects of subthreshold doses of neuromedin (lμg) in the tail-flick test. Maximal antinociception was obtained by combining both inhibitors, thus conferring antinociceptive effects to neuromedin doses that were as low as 10 ng. Naloxone (0.5–2 mg/kg, s.c.) did not significantly reduced the antinociceptive effects of combinations of neurotensin and thiorphan and of neuromedin, thiorphan and bestatin. The data show that both neurotensin and neuromedin elicit analgesia in mice through an opiate independent mechanism. Furthermore, like enkephalin, neuromedin is readily degraded by brain endopeptidase 24.11 and bestatin sensitive aminopeptidase(s), whereas the resistance of neurotensin to aminopeptidase attack confers to this peptide a broader spectrum and longer duration of action than its congener neuromedin.