Differential processing of the neurotensin/neuromedin N precursor in the mouse

Posttranslational processing of the neurotensin/neuromedin N (NT/NN) precursor has been investigated in mouse brain and small intestine by means of region-specific radioimmunoassays coupled to chromatographic fractionations. In brain, total NT/NN immunoreactivity measured with a common C-terminal antiserum was 15.72 pmol/g. NT measured with an N-terminal antiserum was 9.74 pmol/g and NN measured with an N-terminal antiserum was 5.98 pmol/g. In small intestine, combined NT/NN immunoreactivity was 108.55 pmol/g, consisting of 66.37 pmol/g NT but only 0.96 pmol/g NN. Gel permeation chromatography and reverse phase HPLC revealed that the large discrepancy in the NT and NN values obtained in small intestinal extracts was due to the presence of a high molecular weight, hydrophobic peptide, which was reactive only with the common C-terminally directed antiserum. Pepsinization of this generated an immunoreactive peptide with similar chromatographic characteristics to NN. In mouse intestine, NN is only partially cleaved from the common NT/NN precursor, resulting in the presence of an N-terminally extended molecular species. This novel molecular species of neuromedin N may be the physiological mediator of certain peripheral biological effects hitherto attributed to neurotensin or neuromedin N.     

Isolation and structural determination of rat neuromedin U

Rat neuromedin U was isolated from the small intestine using mainly immunoaffinity chromatography and radioimmunoassay for pig neuromedin U-8. The amino acid sequence of rat neuromedin U was determined by microsequence analysis to be Tyr-Lys-Val-Asn-Glu-Tyr-Gln-Gly-Pro-Val-Ala-Pro-Ser-Gly-Gly- Phe-Phe-Leu-Phe-Arg-Pro-Arg-Asn-NH2, and this structure was confirmed by synthesis. Although the C-terminal heptapeptide amide structure of pig neuromedin U is completely conserved in rat neuromedin U, the remainder of the peptide reveals nine amino acid replacements and two amino acid deletions when compared to pig neuromedin U-25. Rat neuromedin U exerts two-fold potent uterus stimulant activity as compared to pig neuromedin U-25.     

Projections of neurons with neuromedin U-like immunoreactivity in the small intestine of the guinea-pig

Summary Neuromedin U immunoreactivity was located histochemically in the guinea-pig small intestine. Projections of immunoreactive neurons were determined by analysing patterns of degeneration following nerve lesions. The co-localization of neuromedin U immunoreactivity with immunoreactivity for substance P, neuropeptide Y, vasoactive intestinal peptide and calbindin was also investigated. Neuromedin U immunoreactivity was found in nerve cells in the myenteric and submucous plexuses and in nerve fibres in these ganglionated plexuses, around submucous arterioles and in the mucosa. Reactive fibres did not supply the muscle layers. Most reactive nerve cells in the myenteric ganglia had Dogiel type-II morphology and in many there was co-localization of calbindin, although some Dogiel type-II neuromedin U neurons were calbindin negative. Lesion studies suggest that these myenteric neurons project circumferentially to local myenteric ganglia. Projections from myenteric neurons also run anally in the myenteric plexus, while other projections extend to submucous ganglia, and still further projections run from the intestine to provide terminals in the coeliac ganglia. In the submucous ganglia neuromedin U was co-localized in three populations of nerve cells: (i) those with vasoactive intestinal peptide immunoreactivity, (ii) neurons containing neuropeptide Y, and (iii) neurons containing substance P. Each of these populations sends nerve fibres to the mucosa. Neuromedin U immunoreactivity is thus located in a variety of neurons serving different functions in the intestine and therefore probably does not have a single role in intestinal physiology.     

Isolation, structural characterization and pharmacological activity of dog neuromedin U

The neuromedin U-like immunoreactivity in an extract of dog small intestine was resolved by reversed-phase HPLC into two molecular forms. The primary structure of the larger form (NMU-25) was established as: Phe-Arg-Leu-Asp-Glu-Glu-Phe-Gln-Gly-Pro10-Ile-Ala-Ser-Gln-Val-Arg- Arg-Gln-Phe- Leu20-Phe-Arg-Pro-Arg-Asn-NH2. This sequence shows five substitutions relative to pig neuromedin U-25. The primary structure of the second peptide (NMU-8) was established as: pGlu-Phe-Leu-Phe-Arg-Pro-Arg-Asn-NH2. The sequence contains the substitution pGlu for Tyr1 compared with pig neuromedin U-8. The potency of synthetic dog NMU-8 in contracting smooth muscle from the rat uterus (EC50 10 +/- 2 nM; mean +/- S.E., n = 6) was not significantly different from the corresponding potency of pig NMU-8 (EC50 16 +/- 5 nM) but the maximum response produced by the dog peptide was greater (58%; p less than 0.05) than that produced by pig NMU-8.     

Neuromedin B-like peptides in rat brain: biochemical characterization, mechanism of release and localization in synaptosomes

Neuromedin B-like peptides were characterized in the rat brain. A rabbit antisera was utilized which recognized neuromedin B but not bombesin or GRP. Using gel filtration and HPLC techniques, a major and minor peak of immunoreactivity was present in rat brain extracts. In both cases the main peak of immunoreactivity coeluted with synthetic neuromedin B. The density of neuromedin B-like peptides ranged 50-fold being greatest in the olfactory bulb and hypothalamus, intermediate in the hippocampus, spinal cord, medulla/pons, pituitary, midbrain, thalamus, striatum and cortex and lowest in the cerebellum. Release studies indicated that neuromedin B-like peptides were secreted from hypothalamic, olfactory bulb and thalamic slices in a Ca++-dependent manner when KCl (75 mM) was present. Also, the neuromedin B-like peptides in the rat brain were localized to synaptosomes. These data indicate that neuromedin B-like peptides may function as regulatory peptides in the CNS distinct from bombesin/GRP.     

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.     

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.     

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.     

The rat gene encoding neurotensin and neuromedin N. Structure, tissue-specific expression, and evolution of exon sequences

Recombinant DNA clones encoding the neurotensin/neuromedin N precursor protein have been isolated from both bovine hypothalamus cDNA and rat genomic libraries using a heterologous canine cDNA probe. Nucleotide sequence analysis of these clones and comparison with the previously determined canine sequence has revealed that 76% of the amino acid residues are conserved in all three species. The protein precursor sequences predicted from bovine hypothalamus and canine intestine cDNA clones vary at only 9 of 170 amino acid residues suggesting that within a species identical precursors are synthesized in both the central nervous system and intestine. The rat gene spans approximately 10.2 kilobases (kb) and is divided into four exons by three introns. The neurotensin and neuromedin N coding domains are tandemly positioned on exon 4. RNA blot analysis has revealed that the rat gene is transcribed to yield two distinct mRNAs, 1.0 and 1.5 kb in size, in all gastrointestinal and all neural tissues examined except the cerebellum. There is a striking variation in the relative levels of these two mRNAs between brain and intestine. The smaller 1.0-kb mRNA greatly predominates in intestine while both mRNA species are nearly equally abundant in hypothalamus, brain stem, and cortex. Sequence comparisons and RNA blot analysis indicate that these two mRNAs result from the differential utilization of two consensus poly(A) addition signals and differ in the extent of their 3' untranslated regions. The relative combined levels of the mRNAs in various brain and intestine regions correspond roughly with the relative levels of immunologically detectable neurotensin except in the cerebral cortex where mRNA levels are 6 times higher than anticipated.     

High-affinity receptors for bombesin-like peptides in normal guinea pig lung membranes.

The binding of the radiolabelled bombesin analogue [125I-Tyr4]bombesin to guinea-pig lung membranes was investigated. Binding of [125I-Tyr4]bombesin was specific, saturable, reversible and linearly related to the protein concentration. Scatchard analysis of equilibrium binding data at 25 degrees C indicated the presence of a single class of non-interacting binding sites for bombesin (Bmax = 7.7 fmol/mg protein). The value of the equilibrium dissociation constant (KD = 90 pM) agrees with a high-affinity binding site. Bombesin and structurally related peptides such as [Tyr4]bombesin, neuromedin B and neuromedin C inhibited the binding of [125I-Tyr4]bombesin in an order of potencies as follows: [Tyr4]bombesin greater than bombesin greater than or equal to neuromedin C much greater than neuromedin B. These results indicate that guinea-pig lung membranes possess a single class of bombesin receptors with a high affinity for bombesin and a lower one for neuromedin B.     

Primary Structure and Tissue Distribution of Guinea Pig Gastrin-Releasing Peptide

The primary structure of gastrin-releasing peptide from the guinea pig stomach has been determined by automated Edman degradation and shown to be identical to porcine gastrin-releasing peptide. Extracts of guinea pig brain and small intestine contained both gastrin-releasing peptide and its COOH-terminal decapeptide (neuromedin C) but the stomach extracts contained only gastrin-releasing peptide. Within the small intestine, highest concentrations of gastrin-releasing peptide-like immunoreactivity were found in extracts of the circular and longitudinal smooth muscle layers.     

Structural identification, subcellular localization and secretion of bovine adrenomedullary neuromedin C [GRP-(18-27)

Bombesin-like immunoreactivity (BLI) was purified from acid (HCl) extracts of bovine adrenal medulla. High performance liquid chromatography (HPLC) on a mu-Bondapak C18 column revealed the presence of five molecular forms of BLI, one coeluting with synthetic gastrin releasing peptide (GRP), the mammalian counterpart of amphibian bombesin, one coeluting with neuromedin C, one coeluting with neuromedin B and the two other ones coeluting with the oxidized forms of neuromedins B and C. The material corresponding to neuromedin C was purified to homogeneity and its amino acid composition and sequence corresponded to those expected for neuromedin C. HPLC analysis on an analytical SP-5PW column of subcellular extracts of bovine adrenal medulla indicated that neuromedin C is almost exclusively localized in secretory granules. The neuropeptide function of neuromedin C and/or other BLI peptides at this level was supported by the stimulatory effect of carbamylcholine (500 microM) on the release of BLI (4.5-fold increase over the basal release of 19 fmol/5 min) from perfused bovine adrenal glands.     

Pharmacologic characterization and autoradiographic distribution of binding sites for iodinated tachykinins in the rat central nervous system

P-type, E-type, and K-type tachykinin binding sites have been identified in the mammalian CNS. These sites may be tachykinin receptors for which the mammalian neuropeptides substance P, neuromedin K, and substance K are the preferred natural agonists, respectively. In the present investigation, we have compared the pharmacology and the autoradiographic distribution of CNS binding sites for the iodinated (¹²⁵I-Bolton-Hunter reagent) tachykinins substance P, eledoisin, neuromedin K, and substance K. Iodinated eledoisin and neuromedin K exhibited an E-type binding pattern in cortical membranes. Iodinated eledoisin, neuromedin K, and substance K each labeled sites that had a similar distribution but one that was considerably different from that of sites labeled by iodinated substance P. CNS regions where there were detectable densities of binding sites for iodinated eledoisin, neuromedin K, and substance K and few or no sites for iodinated substance P included cortical layers IV–VI, mediolateral septum, supraoptic and paraventricular nuclei, interpeduncular nucleus, ventral tegmental area, and substantia nigra pars compacta. Binding sites for SP were generally more widespread in the CNS. CNS regions where there was a substantial density of binding sites for iodinated substance P and few or no sites for iodinated eledoisin, neuromedin K, and substance K included cortical layers I and II, olfactory tubercle, nucleus accumbens, caudate-putamen, globus pallidus, medial and lateral septum, endopiriform nucleus, rostral thalamus, medial and lateral preoptic nuclei, arcuate nucleus, dorsal raphe nucleus, dorsal parabrachial nucleus, parabigeminal nucleus, cerebellum, inferior olive, nucleus ambiguus, retrofacial and reticular nuclei, and spinal cord autonomic and somatic motor nuclei. In the brainstem, iodinated substance P labeled sites in both sensory and motor nuclei whereas iodinated eledoisin, neuromedin K, and substance K labeled primarily sensory nuclei. Our results are consistent with either of two alternatives: (1) that iodinated eledoisin, neuromedin K, and substance K bind to the same receptor site in the rat CNS, or (2) that they bind to multiple types of receptor sites with very similar distribution.     

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.     

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).     

Neuropeptide-γ: A Peptide Isolated from Rabbit Intestine that Is Derived from γ-Preprotachykinin

The neurokinin A-like immunoreactivity in an extract of rabbit small intestine was resolved into two molecular forms by gel permeation chromatography. These components were purified to apparent homogeneity by reverse-phase HPLC. The primary structure of the larger component was established as the following: Asp-Ala-Gly-His-Gly-Gln-Ile-Ser-His-Lys-Arg-His-Lys-Thr-Asp-Ser-Phe-Val- Gly-Leu - Met.NH2. This amino acid sequence represents residues (72-92) of gamma-preprotachykinin, as predicted from the nucleotide sequence of a cloned cDNA from the rat. The peptide, termed neuropeptide-gamma, lacks residues (3-17) of neuropeptide K, and this segment is specified exactly by exon 4 in the preprotachykinin gene. The smaller form of neurokinin A-like immunoreactivity was identical to neurokinin A. Neuropeptide K was not present in the extract, demonstrating that the pathways of post-translational processing of beta- and gamma-preprotachykinins in the rabbit gut are different.     

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.     

Apolipoprotein gene expression in the rabbit: abundance, size, and distribution of apolipoprotein mRNA species in different tissues

We have used human apolipoprotein cDNAs as hybridization probes to study the relative abundance and distribution of apolipoprotein mRNAs in rabbit tissues by RNA blotting analysis. The tissues surveyed included liver, intestine, lung, pancreas, spleen, stomach, skeletal muscle, testis, heart, kidney, adrenal, aorta, and brain. We found that liver is the sole or major site of synthesis of apoA-II, apoA-IV, apoB, apoC-I, apoC-II, apoC-III, and apoE, and the intestine is a major site of synthesis of apoA-I, apoA-IV, and apoB. Minor sites of apolipoprotein mRNA synthesis were as follows: apoA-I, liver and skeletal muscle; apoA-IV, spleen and lung; apoB, kidney; apoC-II and apoC-III, intestine. ApoE mRNA was detected in all tissues surveyed with the exception of skeletal muscle. Sites with moderate apoE mRNA (10% of the liver value) were lung, brain, spleen, stomach, and testis. All rabbit mRNAs had forms with sizes comparable to their human counterparts. In addition, hybridization of hepatic and intestinal RNA with human apoA-IV and apoB probes produced a second hybridization band of approximately 2.4 and 8 kb, respectively. Similarly, hybridization of rabbit intestinal RNA with human apoC-II produced a hybridization band of 1.8 kb. The 8 kb apoB mRNA form may correspond to the apoB-48 mRNA, whereas the apoA-IV- and apoC-II-related mRNA species have not been described previously. This study provides a comprehensive survey of the sites of apolipoprotein gene expression and shows numerous differences in both the abundance and the tissue distribution of several apolipoprotein mRNAs between rabbit and human tissues. These findings and the observation of potentially new apolipoprotein mRNA species are important for our understanding of the cis and trans acting factors that confer tissue specificity as well as factors that regulate the expression of apolipoprotein genes in different mammalian species.