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 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.     

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.     

Altered processing of the neurotensin/neuromedin N precursor in PC2 knock down mice: a biochemical and immunohistochemical study

Neurotensin (NT) and neuromedin N (NN) are generated by endoproteolytic cleavage of a common precursor molecule, pro-NT/NN. To gain insight into the role of prohormone convertases PC1, PC2, and PC7 in this process, we investigated the maturation of pro-NT/NN in the brain of PC7 (PC7-/-), PC2 (PC2-/-), and/or PC1 (PC1+/- and PC2-/-; PC1+/-) knock down mice. Inactivation of the PC7 gene was without effect, suggesting that this convertase is not involved in the processing of pro-NT/NN. By contrast, there was a 15% decrease in NT and a 50% decrease in NN levels, as measured by radioimmunoassay, in whole brain extracts from PC2 null as compared with wild type mice. Using immunohistochemistry, we found that this decrease in pro-NT/NN maturation products was uneven and that it was most pronounced in the medial preoptic area, lateral hypothalamus, and paraventricular hypothalamic nuclei. These results suggest that PC2 plays a critical role in the processing of pro-NT/NN in mouse brain and that its deficiency may be compensated to a regionally variable extent by other convertases. Previous data have suggested that PC1 might be subserving this role. However, there was no change in the maturation of pro-NT/NN in the brain of mice in which the PC1 gene had been partially inactivated, implying that complete PC1 knock down may be required for loss of function.     

Lithium dramatically potentiates neurotensin/neuromedin N gene expression

Lithium perturbs intracellular signal transduction pathways used by neurotransmitters, suggesting that changes in receptor signalling may underlie its actions in the treatment of manic depressive illness. Little attention, however, has been directed toward possible additional actions at the level of specific gene expression, particularly of genes encoding neurotransmitters or neuromodulators. In PC12 pheochromocytoma cells, lithium dramatically potentiates increases in intracellular levels of the neuropeptide neurotensin and the mRNA encoding it, caused by combinations of nerve growth factor, dexamethasone, and the adenylate cyclase activator, forskolin. This result demonstrates that lithium can profoundly influence the expression of a specific neuropeptide gene in a previously unanticipated manner and suggests that changes in gene expression might be involved in its therapeutic activity.     

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.     

Site-specific DNA methylation contributes to neurotensin/neuromedin N expression in colon cancers

The neurotensin/neuromedin N (NT/N) gene is expressed in fetal colon, repressed in newborn and adult colon, and reexpressed in approximately 25% of colon cancers. Our purpose was to determine the effect of gene methylation on NT/N silencing in colon cancers. We found that the NT/N gene was expressed in human colon cancer cell line KM12C but not in KM20 colon cancer cells. Bisulfite genomic sequencing demonstrated that all CpG dinucleotides in the region from -373 to +100 of the NT/N promoter, including a CpG site in a distal consensus AP-1 site, were methylated in KM20 but unmethylated in KM12C cells. Treatment of KM20 cells with demethylating agent 5-azacytidine induced NT/N expression, suggesting a role for DNA methylation in silencing of NT/N in colon cancers. To better elucidate the mechanisms responsible for NT/N repression by DNA methylation, we performed gel shift assays using an oligonucleotide probe corresponding to the distal AP-1 consensus sequence of the NT/N promoter. Methylation of the oligonucleotide probe inhibited protein binding to the distal AP-1 site of the NT/N promoter, suggesting a potential mechanism of NT/N gene repression in colon cancers. We show that DNA methylation plays a role in NT/N gene silencing in the human colon cancer KM20 and that NT/N expression in KM12C cells is associated with demethylation of the CpG sites. DNA methylation likely contributes to NT/N gene expression noted in human colon cancers.     

Receptors for neurotensin in canine small intestine.

We have previously characterized the neurotensin receptors on the circular smooth muscle (CM) of the canine small intestine (1). In the present studies, using radioligand binding technique, neurotensin receptors were localized on the membranes from deep muscular (DMP) and the submucous plexus while no binding was observed on either the longitudinal smooth muscle or myenteric plexus membranes. The high affinity binding sites (Kd 0.1-0.2 nM) on DMP membranes were similar to those on CM; the low affinity component was of much lower affinity (Kd approximately 40 nM). DMP had 4-6 times higher density of binding sites than the CM. The recognition properties of DMP receptors were similar to those on the CM and reduced sulfhydryl groups were required for the binding activity. The action of neurotensin on the contractility of the canine small intestine, therefore, appears to be through a direct action on the circular smooth muscle and through the prejunctional action on the DMP neurons through distinct receptors. Thiol groups in the neurotensin receptors may be important for the receptor function.     

A comparison of the effects of mesencephalic injections of neurotensin(1-13) and neuromedin N on brain electrical self-stimulation.

Neuromedin N (NM-N), a hexapeptide that shares a four amino acid C-terminal homology with the tridecapeptide, neurotensin (NT), has been suggested as a potential neurotransmitter or neuromodulator that could interact with the NT-sensitive receptors. In this experiment, we compared the effects of an equimolar concentration of NM-N and NT(1-13) injected in the ventral tegmental area (VTA) on brain electrical self-stimulation (SS), a behavior previously shown to be potentiated by VTA injections of NT(1-13). Rats implanted with a stimulating electrode in the mesencephalic central gray and a guide cannula in the VTA were trained to lever press to obtain rewarding electrical stimulations. Functions relating the rate of lever pressing to the stimulation frequency were determined, on separate daily tests, before and after the injection of 3 nmol of NM-N, NT(1-13), or an equal volume of saline vehicle. At this concentration, both NM-N and NT(1-13) produced a significant facilitation of SS when compared to saline vehicle, an effect that was not seen when the peptides were injected outside the VTA. The facilitation of SS by NM-N, however, was much weaker and of a shorter duration than the one produced by NT(1-13). The shorter time course and the weaker behavioral effect of NM-N compared to NT(1-13) are consistent with its lower potency at the NT receptor and its faster rate of enzymatic degradation in the VTA, and suggest that NM-N potentiated the reward-relevant neural signal by acting on mesencephalic NT receptors.     

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.     

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.     

Modulation by neurotensin and neuromedin N of adherence and chemotaxis capacity of murine lymphocytes.

The action of neurotensin and related peptides has not been yet studied on lymphocytes, although there are studies indicating the stimulative action of neurotensin, a peptide first isolated from bovine hypothalamus, on different functions of phagocytic immune cells. The present study demonstrates that neurotensin and a related peptide, neuromedin N, increased significantly the adherence and chemotaxis capacity of murine peritoneal lymphocytes, when they were incubated in the presence of neuropeptide concentrations between 10(-9) M and 10(-12) M. With respect to their adherence capacity, neuromedin N showed a slightly higher stimulation than neurotensin at a shorter time. However, both neuropeptides stimulated the chemotaxis capacity in a similar percentage. The study of the action mechanisms of these neuropeptides showed that intracellular cAMP levels were not modified by neurotensin or neuromedin N, but using an extracellular calcium chelator, EGTA (1 mM), and a blocker of calcium channels in endoplasmic reticulum, ryanodine (0.5 mM), we observed that neurotensin and neuromedin N could produce their effects through an augmentation of the intracellular Ca2+ concentration. As adherence and chemotaxis are initial processes of immune response, the results show that both neuropeptides may be physiological modulators of the lymphocyte function.     

A protein kinase inhibitor, staurosporine, mimics nerve growth factor induction of neurotensin/neuromedin N gene expression.

Nerve growth factor (NGF) cooperates with glucocorticoids, activators of adenylate cyclase, and lithium to induce the expression of teh gene encoding the neuropeptides neurotensin and neuromedin N (NT/N gene) in PC 12 pheochromocytoma cells. High level expression requires simultaneous treatment with three or all four inducers. To examine the mechanism underlying this complex synergism, we have examined the effects of protein kinase inhibitors and other agents which influence intracellular signal transduction on NT/N gene expression. Two structurally similar bacterial alkaloids, staurosporine and K-252a, inhibit several protein kinases in vitro, including protein kinase C and cyclic nucleotide-dependent kinases. K-252a has been reported to specifically inhibit the effects of NGF on PC12 pheochromocytoma cells. Surprisingly, staurosporine in combination with other inducers markedly potentiated NT/N gene expression. In contrast, K-252a had no effect on NT/N gene expression when added simultaneously with other inducers. Expression of the NT/N gene was also potentiated by the phorbol ester 12-O-tetradecanoylphorbol-13-acetate, which directly activates protein kinase C, and by bradykinin, which stimulates phosphatidylinositol turnover in PC12 cells, and these effects were not blocked by staurosporine. Staurosporine was generally more effective in stimulating NT/N gene expression when used in inducer combinations that did not include NGF. These results, taken together with recent evidence that staurosporine is also able to induce neurite outgrowth from PC12 cells, suggest that the effects of staurosporine and NGF may converge, in part, on a common intracellular target.     

Characterization of promoter elements required for cell-specific expression of the neurotensin/neuromedin N gene in a human endocrine cell line.

Expression of the gene encoding neurotensin/neuromedin N (NT/N) is mostly limited to the brain and specialized enteroendocrine cells (N cells) of the distal small intestine. We have analyzed the NT/N DNA sequences upstream of the RNA start site that direct cell-specific expression using a novel human endocrine cell line, BON, that resembles intestinal N cells in several important aspects, including NT/N precursor protein processing, ratios of different NT/N mRNA forms, and high levels of constitutive expression of the NT/N gene. Transient transfection assays with plasmids with progressive 5' deletions of the rat NT/N promoter identified the proximal 216 bp of 5' flanking sequences as essential for high-level constitutive NT/N expression in BON cells. In addition, a detailed mutational analysis defined multiple regions within the proximal 216 bp that contribute to cell-specific NT/N expression. These elements include a proximal cyclic AMP response element (CRE)/AP-1-like motif (TGACATCA) that binds c-Jun, JunD, CRE-binding (CREB), and ATF proteins, a near-consensus glucocorticoid response element, and a distal consensus AP-1 site that binds c-Fos, Fra-1, and JunD. In addition, elements contained within two 21-bp imperfect direct repeats play an important role in NT/N expression in BON cells and may bind novel factors that act as positive regulators of NT/N expression. DNase I footprinting and gel shift analyses demonstrate that the sites identified by mutational analysis, and at least one additional site, specifically bind BON cell nuclear proteins in vitro. We speculate that a complex pattern of regulation requiring interaction between a proximal CRE/AP-1-like motif and other upstream control elements play an important role in the high-level constitutive expression of NT/N in the human endocrine cell line BON. In addition, the BON cell line provides a unique model to further characterize the factors regulating cell-specific NT/N expression and to better understand the mechanisms responsible for the terminal differentiation of the N-cell lineage in the gut.     

A common precursor to neurotensin and LANT6 and its differential processing in chicken tissues

Antisera towards neurotensin (NT) and the structurally related peptide, LANT6, were used to characterize immunoreactive peptides and proteins in extracts of chicken tissues. A 17 kDa protein was identified by Western blotting as a potential precursor to NT and LANT6. However, the posttranslational processing of this common precursor appeared to be tissue specific, giving rise to disproportionate amounts of NT and LANT6, along with varying expression of a large molecular LANT6 (M_r, 15 kDa). The intestinal cells containing immunoreactive NT, LANT6, and large molecular LANT6 behaved similarly during fractionation by size and density. These activities also banded together in particles resembling vesicles during centrifugation of isotonic homogenates of tissue. These results suggest that chicken NT and LANT6 are biosynthesized as parts of the same precursor, the processing of which can give rise to a variety of products stored within secretory vesicles.     

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.