Portion Of Neurotensin Research Articles

A Simple Test To Detect Hydrogen/Deuterium Scrambling during Gas-Phase Peptide Fragmentation

Amide hydrogen/deuterium (H/D) exchange coupled with mass spectrometry has become a powerful tool to study protein dynamics. Addition of a proteolysis step between the exchange reaction and mass analysis can be used to localize the positions of deuterium and improve overall resolution. The resolution can be further enhanced by the fragmentation of digested peptides in the gas phase if scrambling of exchangeable hydrogens and deuteriums on the peptides does not occur. Although some laboratories reported successful localization of deuteriums by gas-phase fragmentations, others described total scrambling. Here we propose a simple method to detect the presence or absence of scrambling using a commercially available small peptide, neurotensin (9-13; RPYIL). All exchangeable hydrogens on this pentapeptide are first deuterated by dissolving it in deuterium oxide. The deuterated peptide is loaded onto a reversed-phase column, and then washed with copious amounts of cold acidic aqueous buffer. This washing exchanges all deuteriums on both the terminals and the side chains back to hydrogens. Now only three deuteriums are attached on the pentapeptide, one on each of the amide nitrogens of Y, I, and L. After the partially deuterated peptide is eluted from the column with 95% acidic acetonitrile, collision-induced dissociation (CID) generates a series of b ions, which are analyzed by mass spectrometer. In the absence of scrambling, no deuterium should be observed in the b 2 ion, as neither R nor P have amide hydrogens. On the other hand, in the event of scrambling, b 2 should carry about half of the deuteriums of the parent pentapeptide. In theory, complete scrambling should distribute deuteriums equally among all of the exchangeable hydrogens. The b 2 portion of neurotensin (9-13) has 6 exchangeable hydrogens, whereas the +1 charge state of neurotensin (9-13) has 12 exchangeable hydrogens. We demonstrated that CID caused complete scrambling of hydrogens and deuteriums with an LCQ (a ion trap machine).

Reduced amide bond neurotensin 8–13 mimetics with potent in vivo activity

Appropriately substituted 8–9 (ΨCH 2NH) isosteres of neurotensin (NT) 8–13 have been found which are active as NT agonists in vitro and in vivo. SAR studies suggest that preventing amide bond hydrolysis at the 8–9 and 11–12 positions of NT(8–13) mimetics is important for producing compounds with potent activity in vivo. Other simplified replacements for the Arg-Arg portion of NT(8–13) are reported.

Chicken liver contains a large quantity of a G-protein-linked neurotensin receptor.

Using 125I-labeled neurotensin (NT), chicken liver was found to contain high affinity, G-protein-linked receptors directed specifically towards the bioactive C-terminal portion of NT. Binding was proportional to membrane and optimal at pH 7.5. The apparent Kd (approximately 91 pM) for this single class of binding sites was similar to Kds reported for the high-affinity components of NT binding to mammalian brain and intestinal membranes. However, the binding capacity (Bmax, approximately 2.3 pmol/mg) was 10-100 times higher than values reported for these mammalian tissues. Binding was inhibited by GTP analogues and by treatment with pertussis toxin but not by cholera toxin. Treatments with alkaline solutions, shown to inactivate G-proteins, decreased subsequent binding at pH 7.5. Whereas low concentrations of Mg2+ (optimum, approximately 0.5 mM) enhanced NT binding, concentrations of 5 mM and above were inhibitory. Cross-linking of 125I-labeled NT to liver membranes using glutaraldehyde specifically labeled two substances of approximately 52 and approximately 90 kDa, which could represent different binding proteins or complexes. These data demonstrate the presence in chicken liver of large amounts of high-affinity NT receptor(s) coupled to pertussis toxin-sensitive G-protein(s).

Vasoactive intestinal peptide potentiates sexual behavior: inhibition by novel antagonist.

Vasoactive intestinal peptide (VIP) has been suggested as a neurotransmitter mediating penile erection. We now show that VIP can stimulate sexual behavior in rats with reduced masculine potential due to pituitary grafting or castration. This effect was attenuated in the presence of a novel VIP antagonist, devised by a hybrid peptide strategy. Thus, we have synthesized a molecule combining a portion of VIP with a portion of neurotensin, peptides of opposite pharmacological action on cAMP formation and smooth muscle relaxation. The hybrid peptide markedly inhibited VIP's effect on sexual behavior. This inhibition was manifested by a significant increase in the mean interval between copulatory events (greater than 3-fold change) coupled with a blockade of VIP-stimulated ejaculation. Other putative VIP antagonists were not as effective in blocking these activities. Thus, our results imply that VIP is not only associated with penile erection, but is involved in sexual behavior as well. Furthermore, the hybrid antagonist was shown to inhibit VIP binding in glial cell cultures. The availability of highly potent VIP antagonists may offer a route to study the possible multiple VIP receptors as well as help delineate other biological activities attributable to VIP.

Effect of synthetic neuromedin-N, a novel neurotensin-like peptide, on exocrine pancreatic secretion and splanchnic blood flow in dogs

Neuromedin-N, a novel neurotensin-like peptide, has recently been identified from porcine spinal cord by using a bioassay for a stimulatory effect on guinea pig ileum. The amino acid sequence of the peptide has been determined to be Lys-Ile-Pro-Tyr-Ile-Leu, which is quite homologous to the COOH-terminal sequence of neurotensin. In this study, the effect of neuromedin-N on pancreas and splanchnic blood flow was investigated in eight dogs. Intravenous injections of graded doses of synthetic neuromedin-N caused a dose-dependent decrease of systemic arterial pressure and a dose-dependent increase in both portal and superior mesenteric arterial blood flow, which were measured with transit time ultrasonic flow meter. Volume and protein output of the pancreatic juice were also increased significantly by Neuromedin-N. Pancreatic capillary blood flow measured with laser Doppler flowmetry was increased in a dose-related manner. The present study first demonstrated that neuromedin-N retains a potent stimulatory effect on the pancreas and splanchnic circulation, indicating that this peptide is one of the biological active forms of neurotensin-like peptide in mammals. This study also leads to the suggestion that this peptide possesses physiological significance as a novel neuropeptide. Neuromedin-N is a novel hexapeptide which has recently been isolated from porcine spinal cord by using a bioassay for stimulating effect on the contractility of isolated guinea pig ileum by Minamino et al. (1). They have reported that the contractile activity of this paptide is similar to, and estimated to be about 15% of that of neurotensin. They have also reported that the peptide induced a hypotensive response on rat cardiovascular system in the same way as neurotensin (1). To our knowledge the report by Minamino et al. has been the only information available for us regarding the biological activity of neuromedin-N. The influence of neuromedin-N on digestive organs is not known at all. Concerning the structure of neuromedin-N, it has remarkable sequential homology to the COOH-terminal region of neurotensin, and shares its four COOH-terminal amino acids in common with neurotensin (1). Because of the fact that the (8–13) portion of neurotensin is the essential structure for its complete activity (2,3), neuromedin-N is considered to be a minimum and sufficient molecule to exhibit the complete neurotensin-like biological activities. Neuromedin-N has a structural homology also to LANT-6 isolated from chicken intestine (4), and xenopsin isolated from amphibian (5). And these shorter neurotensin-like peptides may constitute a new subclass of neurotensin-like peptide (1) (Table 1). In this study we have investigated the effect of synthetic neuromedin-N on exocrine secretion and capillary blood flow of the pancreas, and on blood flows in portal vein and superior mesenteric artery. Table 1. Amino cid sequence of Neurotensin-kike peptides. Lys-Ile-Pro-Tyr-Ile-Leu Neuromedin-N (procine) pGlu-Leu-Tyr-Glu-Asn-Lys-Pro-Arg-Arg-Pro-Tyr-Ile-Leu Neurotensin (human) Lys-Asn-Pro-Tyr-Ile-Leu LANT-6 (chicken) pGly-Gly-Lys-Arg-Pro-Trp-Ile-Leu Xenopsin (amphibinan)

Pepsin Treatment of Mammalian Plasma Generates Immunoreactive and Biologically Active Neurotensin-Related Peptides in Micromolar Concentrations*

Treatment of mammalian plasmas or sera with pepsin gave rise to extraordinary quantities (1-5 microM) of immunoreactive neurotensin (NT)-related peptide(s). Although immunochemical and chromatographic analyses indicated that the peptides liberated by pepsin differed from authentic neurotensin, one of the major immunoreactive products displayed NT-like biological properties. Partially purified preparations of the immunoreactive peptides increased cutaneous vascular permeability when injected intradermally in rats and released histamine from isolated rat mast cells. The pepsin-generated peptides appeared to share the biologically active C-terminal portion of NT, since they reacted with antisera selective for this region but were not recognized by N-terminal-directed antisera. Gel permeation chromatography demonstrated the presence of two substrates in plasma which liberated iNT upon treatment with pepsin, one in the albumin fraction (mol wt, 65 K) and the other a globulin (mol wt, 350 K). A variety of other proteases and substrates failed to have this effect. These results suggest the existence of a system(s) in blood, analogous to the renin-angiotensin system, for the generation of biologically active NT-related peptides.