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.     

Isolation, biological and chemical characterization, and synthesis of a neurotensin-related hexapeptide from chicken intestine

A new biologically active peptide of the neurotensin (NT) family, shown previously to cross-react in a COOH-terminal-directed radioimmunoassay for bovine NT, has been isolated from extracts of chicken intestine and identified as H-Lys-Asn-Pro-Tyr-Ile-Leu-OH, which is identical with the biologically active COOH-terminal half of NT except for the amino acid substitutions Lys/Arg and Asn/Arg. It is proposed that this peptide be referred to as Lys8, Asn9, NT8-13 (LANT-6). Synthetic material prepared with this amino acid sequence using the Merrifield technique was immunochemically, chromatographically, and biologically indistinguishable from the native peptide. In contrast to chicken NT which induced hypotension, hyperglycemia, increased vascular permeability, and cyanosis when injected intravenously into anesthetized rats, synthetic LANT-6 brought about primarily a hypertensive response and had little ability to promote hyperglycemia, increased vascular permeability, and cyanosis. In rats pretreated with the alpha-blocker phentolamine and in adrenalectomized rats, the hypertensive response to LANT-6 was blocked, suggesting that adrenal catecholamines mediated this effect. These findings suggest that LANT-6, a natural variant of NT with a different spectrum of biologic activity, may be a NT-related messenger peptide with a different function(s).     

Neurotensin elevates hematocrit and plasma levels of the leukotrienes, LTB4, LTC4, LTD4 and LTE4, in anesthetized rats.

The IV injection of neurotensin (NT) into anesthetized rats produced a marked increase in hematocrit, labored breathing and peripheral blood stasis with cyanosis. This effect could also be produced by the NT-related peptides, neuromedin-N and xenopsin; however, it was not observed when nine other biologically active peptides, including bradykinin and substance P, were tested. Associated with these responses were increases in the plasma levels of histamine (measured radioenzymatically) and the leukotrienes, LTB4, LTC4, LTD4, and LTE4 (measured by RIA and HPLC). The increment in hematocrit after varying doses of NT correlated to the increase in plasma levels of LTC4. Histamine and LTC4 were both capable of elevating hematocrit when given IV; however, LTC4 was approximately 1000 times more potent than histamine and active doses of histamine elevated LTC4 levels. Furthermore, the effects of NT on plasma LTC4 and hematocrit were reduced by pretreating animals with antagonists to histamine and serotonin. Pretreatment with the specific mast cell degranulating agent, compound 48/80, also blocked NT's ability to elevate plasma levels of histamine, LTB4 and LTC4 and prevented the increased hematocrit and cyanosis. These results indicate that NT-related peptides are very potent and specific stimulators of leukotriene release and that this action is mediated by mast cells and associated with loss of plasma volume and blood stasis. A working hypothesis is that histamine, released from mast cells in response to NT, stimulates LTC4 production by other cells.     

Plasma levels of human neurotensin: methodological and physiological considerations

The ingestion of a meal high in fat content is known to increase circulating levels of neurotensin (NT) in humans. However, the magnitude of the postprandial rise of NT in the general circulation and its physiological significance have been subject of much debate. The present study examines circulating levels of NT in male volunteers prior to and following each of their three daily meals (ca. 31 g fat/meal). The response observed are also compared to that elicited by the direct instillation of intralipid (ca. 44 g fat) into the duodenum. NT levels were determined by radioimmunoassay of acid/acetone extracted plasma fractionated by high pressure liquid chromatography. Meals caused a significant but modest increase in NT levels, with the largest increment (ca. 4 fmol/ml) occurring after breakfast. In contrast, NT levels increased ca. 20 fmol/ml with intraduodenal instillation of lipid. The meal-stimulated increases in circulating NT measured here are 4- to 5-fold less than those reported by others, the difference most likely reflecting the lesser amount of lipid ingested. Previous studies provided subjects with single meals containing in excess of 120 g of fat; the 30 g of fat ingested by our subjects, ca. 33% of total caloric intake, is near that recommended by the U.S. Senate, Select Committee on Nutritional and Human Needs. These data show that diets with a reasonable fat content have only a modest effect on circulating levels of NT.     

Shedding of the major plasma membrane sialoglycoprotein from the surface of 13762 rat ascites mammary adenocarcinoma cells

ASGP-1 (ascites Sialoglycoprotein 1) the major sialoglycoprotein of 13762 rat ascites mammary adenocarcinoma cells, is shed from MAT-B1 (nonxenotransplantable) and MAT-C1 (xenotransplantable) sublines when incubated in vitro after labeling in vivo with [³H]glucosamine. The rates of shedding of label in both particulate and soluble form are similar for the two sublines, but the turnover of label in the cells is 80% greater for MAT-C1 cells ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ 2.4 days) than for MAT-B1 cells ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ 4.1 days). Shed soluble ASGP-1 was smaller than ASGP-1 in the particulate fraction by gel filtration in dodecyl sulfate. By CsCl density gradient centrifugation, gel filtration, and sucrose density gradient centrifugation, all in 4 m guanidine hydrochloride, the shed soluble ASGP-1 was found to be slightly more dense and smaller than ASGP-1 purified from membranes. No differences in sialic acid or oligosaccharides released by alkaline borohydride treatment were found between the shed soluble ASGP-1 and purified ASGP-1. These results suggest that the shed soluble ASGP-1 is released from the membrane by a proteolytic cleavage. This mechanism is supported by the inhibition of the release of soluble shed ASGP-1 by aprotinin, a protease inhibitor. Soluble ASGP-1 in ascites fluid is also smaller by gel filtration, but is more heterogeneous, suggesting a similar release mechanism in vivo followed by more extensive degradation in the ascites fluid.     

Isolation and partial characterization of ascites sialoglycoprotein-2 of the cell surface sialomucin complex of 13762 rat mammary adenocarcinoma cells.

Sialomucins are the dominant components of the cell surfaces of some carcinoma ascites cells and have been postulated to inhibit recognition of tumours by the immune system. The sialomucin ASGP-1 (ascites sialoglycoprotein-1) of the 13762 rat mammary adenocarcinoma is associated with the cell surface as a complex with a concanavalin-A-binding glycoprotein called ASGP-2. This sialomucin complex has been purified from ascites cell microvilli by extraction with Triton X-100 and CsCl density-gradient centrifugation. ASGP-1 (which has been purified previously) and ASGP-2 were dissociated in 6 M-guanidine hydrochloride and separated by gel filtration. The molecular mass of the undenatured detergent complex of ASGP-2, estimated by gel filtration and velocity sedimentation in Triton X-100, was 148 kDa. Since the apparent molecular mass by SDS/polyacrylamide-gel electrophoresis was about 120 kDa, ASGP-2 must be a monomer as extracted from the membrane. Studies of its chemical composition indicate that it contains about 45% carbohydrate by weight, including both mannose and galactosamine. Alkaline borohydride treatment of ASGP-2 converted approx. half of the N-acetylgalactosamine to N-acetylgalactosaminitol, demonstrating the presence of O-linked oligosaccharides. Analyses of mannose-labelled Pronase glycopeptides from ASGP-2 by lectin-affinity chromatography on concanavalin A and leucocyte-agglutinating phytohaemagglutinin suggested that 40% of the label was present in high-mannose/hybrid oligosaccharides, 20% in triantennary oligosaccharides substituted on the C-2 and C-4 mannose positions and 40% in tri- or tetra-antennary oligosaccharides substituted on C-2 and C-6. The presence of polylactosamine sequences on these oligosaccharides was suggested by lectin blots and by precipitation from detergent extracts with tomato lectin. From chemical analyses and lectin-affinity studies, we estimate that ASGP-2 contains four high-mannose and 13 complex N-glycosylated oligosaccharides, plus small amounts of polylactosamine and O-linked oligosaccharides. The presence of four different classes of oligosaccharides on this glycoprotein suggests that it will be an interesting model system for biosynthetic comparisons of the different glycosylation pathways.     

Species variability in the modification of erythrocyte surface proteins by enzymatic probes

Bovine and equine erythrocytes have been studied by three different surface modification techniques to investigate the accessibility of the surface components to the external medium. Lactoperoxidase labeling of equine erythrocytes results in a significant labeling of only one membrane component, a 100 000-mol.wt polypeptide corresponding to the membrane-spanning Component III of human erythrocytes. The major sialoglycoprotein of the equine erythrocyte is not labeled. This is in contradistinction to the situation for human and bovine cells, where both components are labeled. The equine membrane sialoglycoprotein is also not markedly affected by pronase, chymotrypsin or trypsin treatment of whole cells under the treatment conditions used, although it can be cleaved by pronase in isolated membranes. Experiments with the isolated glycoprotein show that its cleavage by trypsin is quite selective, whereas cleavage by pronase and chymotrypsin is much more extensive. Labelling of bovine red cells by galactose oxidase treatment followed by reduction with ³H-labeled borohydride yields radioactivity in only one major peak, that corresponding to the glycoprotein. Pretreatment of the cells with neuraminidase causes a dramatic increase in the labeling. Equine erythrocytes do not show significant labeling by this technique unless a neuraminidase pretreatment has been performed. Then only the major glycoprotein is labeled. Thus the equine glycoprotein is apparently inaccessible to the cell surface by standard surface modification methods, although it is clearly a surface component. These experiments point out some of the limitations of surface labeling and proteolysis methods in probing the accessibility of membrane components. The results suggest that the apparent inaccessibility of the equine glycoprotein is due partially to its structure and partially to its localization in the membrane.     

Immunohistochemical localization of neurotensin in endocrine cells of the gut

Summary Endocrine cells displaying neurotensin immunoreactivity are found scattered in the jejuno-ileum of all mammals studied, including man. They are rather scarce in rat, guinea pig, rabbit and pig and fairly numerous in cat, dog and man. In most mammals the neurotensin cells predominate on the villi. Only in the dog are they more numerous in the crypts. In the chicken, neurotensin cells occur all along the intestinal tract. They are particularly numerous in the zone that joins the gizzard with the duodenum. The ontogeny of the neurotensin cells in the gut was studied in rats and chickens. In the rat, the cells are first observed in the jejuno-ileum immediately before birth. The adult frequency is reached 4–5 days later. In the chicken, neurotensin cells first appear in the colon in the 18 day old embryo and in the small intestine two days later (i.e. one or two days before hatching). A few days after hatching, the gut has achieved the adult number of neurotensin cells per unit area.