Histochemical and ultrastructural identification of neurotensin cells in the dog ileum

Summary In the dog ileum, neurotensin cells stained with immunofluorescence or immunoperoxidase proved distinct from argentaffin (EC) cells, glucagon immunoreactive (GLI) cells and pancreatic peptide immunoreactive (PP) cells. Neurotensin cells showed various degrees of reactivity with Grimelius'silver. With electron microscopy, besides EC cells, large granule cells with a thin peripheral rim of Grimelius-reactivity (L cells) and large granule cells with variable Grimelius-reactivity of the core (N cells) were found. On distributive grounds, L cells were identified with GLI cells and N cells were interpreted as neurotensin cells.     

Neurotensin Stimulates Inositol Phospholipid Metabolism and Calcium Mobilization in Murine Neuroblastoma Clone N1E-115

Murine neuroblastoma cells (clone N1E-115) possess neurotensin receptors that mediate cyclic GMP synthesis. Because of the hypothesized relationship between phospholipid metabolism, intracellular Ca2+, and cyclic GMP synthesis, we determined with these cells the effects of neurotensin on 32P labeling of phospholipids, release of inositol phosphates, and intracellular Ca2+ (as determined with the use of Quin-2, a fluorescent probe sensitive to free Ca2+ levels). Neurotensin stimulated incorporation of 32P into phospholipids, especially phosphatidylinositol and phosphatidate. Neurotensin also stimulated the release of [3H]inositol phosphates with an EC50 of about 1 nM. Mean basal Ca2+ concentration in these cells was 134 nM and this level was increased in a rapid and dose-dependent manner by neurotensin, with an EC50 of 4 nM. Since the EC50 for neurotensin in stimulating cyclic GMP synthesis is 1.5 nM and the KD for binding of [3H]neurotensin at 0 degrees C is 11 nM, all these different effects appear to be shared proximal consequences of neurotensin receptor activation.     

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.     

Glucose, insulin, pancreatic glucagon and glucagon-like immunoreactive materials in the plasma of normal and diabetic children. Effect of the initial insulin treatment.

Pancreatic glucagon (PG) and other glucagon-like immunoreactive materials (GLI) were measured in the plasma of normal and of newly diagnosed untreated diabetic children, using an antiglucagon serum (AGS) highly specific for pancreatic glucagon (AGS 18) and an AGS which crossreacts with extracts of intestinal mucosa (AGS 10). Gut GLI was considered to be the difference between "total" GLI (AGS 10) and PG (AGS 18). Glucose and immunoreactive insulin (IRI) were also measured. PG, total GLI and gut GLI were significantly elevated in children with severe insulin insufficiency and were reduced to normal by insulin treatment, even though a significant fasting hyperglycemia was still present. In three diabetic children who had high initial plasma IRI levels the three glucagon fractions were normal. We conclude that insulin insufficiency is characterized not only by high plasma levels of PG as previously reported, but also of gut GLI. These abnormalities can be corrected by the administration of insulin.     

Contribution of the neural cell recognition molecule NB‐3 to synapse formation between parallel fibers and Purkinje cells in mouse

The neural cell recognition molecule NB‐3, also referred to as contactin‐6, is expressed prominently in the developing nervous system after birth and its deficiency has been shown to cause impairment in motor coordination. Here, we investigated the contribution of NB‐3 to cerebellar development, focusing on lobule 3 where NB‐3 was expressed in granule cells but not in Purkinje cells. In the developing molecular layer, the neural cell recognition molecules TAG‐1, L1, and NB‐3 formed distinct expression zones from the external granule cell layer to the internal granule cell layer (IGL), respectively. The NB‐3‐immunoreactive zone did not overlap with TAG‐1‐immunoreactive zone. By contrast, the L1‐immunoreactive zone overlapped with both the TAG‐1‐ and NB‐3‐immunoreactive zones. NB‐3‐positive puncta overlapped with vesicular glutamate transporter 1, a presynaptic marker and were apposed close to metabotropic glutamate receptor 1A, a postsynaptic marker, indicating that NB‐3 is localized presynaptically at glutamatergic synapses between parallel fibers and Purkinje cells. In NB‐3 knockout mice, L1 immunoreactive signals were increased in the IGL at postnatal day (P) 5, suggesting the increase in the number of immature granule cells of the IGL. In addition, the density of parallel fiber synaptic terminals was reduced in NB‐3 knockout mice relative to wild‐type mice at P5 to P10. In parallel with these findings, caspase‐dependent cell death was significantly increased in the NB‐ 3‐deficient cerebellum at P15. Collectively, our results indicate that NB‐3 deficiency affects synapse formation during postnatal cerebellar development. © 2009 Wiley Periodicals, Inc. Develop Neurobiol, 2009     

Distribution patterns of neurotensin-like immunoreactive cells in the gastro-intestinal tract of higher vertebrates

Summary The endocrine system of the gastro-intestinal tract of selected species representing the five higher vertebrate classes was investigated with reference to occurrence and distribution of neurotensin-like immunoreactive cells. Using antibodies against C-terminal and N-terminal fragments of neurotensin and against the C-terminal sequence of xenopsin it was demonstrated that the intestine of all species studied contains endocrine, neurotensin-like immunoreactive cells. However, large differences in localization and frequency of these neurotensin-like immunoreactive cells were found. Except for a teleostean fish, neurotensin-like immunoreactive cells in the gastro-intestinal tract were more frequent in non-mammalian vertebrates than in mammals. In contrast to mammals, where the highest density of neurotensin-like immunoreactive cells was present in the ileal mucosa, in the non-mammalian vertebrates studied the corresponding cells were most abundant in the pyloric-duodenal junction. The exact mapping of neurotensin-like immunoreactive cells is presented throughout the entire gastro-intestinal tract of six species (Rattus, Coturnix, Lacerta, Rana, Xenopus, Carassius) including a quantitative evaluation of sequential serial sections.     

Immunohistochemical localization of some endocrine cells in the gastroenteropancreatic system of Erinaceus europaeus

The distribution of chromogranin A and neuron specific enolase (NSE) in the neuroendocrine gut system and the morphology and distribution of cells containing gastrin, somatostatin, neurotensin and VIP in the gastroenteropacreatic (GEP) apparatus of Erinaceus europaeus were investigated by immunohistochemical methods. Chromogranin A and somatostatin immunoreactive cells were present throughout the gastrointestinal mucosa, with the exception of the oesophagus and in the pancreas. Gastrin cells were peculiar of the pyloric glands and duodenal mucosa and neurotensin cells of the small intestine. No VIP immunoreactive endocrine cells were noticed in the GEP system. VIP and NSE immunoreactivities were detected both in nerve cell bodies and terminals of the wall of the GEP apparatus. NSE immunoreactivity was found in the endocrine cells of the fundic and pyloric mucosa.     

The Rat Neurotensin Receptor Expressed in Chinese Hamster Ovary Cells Mediates the Release of Inositol Phosphates

To study second messenger synthesis mediated by the cloned rat neurotensin receptor, we derived a cell line stably expressing this receptor. The cDNA clone of this receptor was subcloned into the pcDNA1neo expression vector. This construct was then used to transfect Chinese hamster ovary (CHO)-K1 cells. Colony clones, selected for resistance to antibiotic G-418 sulfate, were isolated and grown separately. Nineteen individual clones were screened for total [3H]neurotensin binding as an indication of neurotensin receptor expression. The clone (CHO-rNTR-10) showing the highest level of specific [3H]neurotensin binding was characterized further. With intact cells, the equilibrium dissociation constant (KD) for specific [3H]neurotensin binding was 18 nM, and the maximal number of binding sites (Bmax) was 900 fmol/mg of protein or 740 fmol/10(6) cells (approximately 4.4 x 10(5) sites on the cellular surface). Whereas the KD was similar to that found in other cellular systems, for example, the murine neuroblastoma clone N1E-115, the Bmax exceeded previously reported values. Incubation of intact CHO-rNTR-10 cells with neurotensin caused the release of inositol phosphates in a dose-dependent manner (EC50 = 3 nM), results indicating that the expressed transfected receptor was functional. Neurotensin did not inhibit cyclic AMP levels stimulated by forskolin. As with other systems, neurotensin (8-13) was more potent than neurotensin Neurotensin-mediated inositol phosphate release is the first report of second messenger synthesis for this receptor expressed in a transfected cell line. These results suggest that the relation between structure and function of the neurotensin receptor can be readily studied in transfected cell lines.     

Activation of B2 bradykinin receptors by neurotensin

Many previous reports suggested that relatively high concentrations of neurotensin were required to exert its effects on neurotransmitter secretion. The neurotensin binding sites, which recognize high concentrations of neurotensin, were characterized in rat pheochromocytoma (PC12) cells. When PC12 cells were treated with neurotensin, [3H]norepinephrine secretion and elevation of cytosolic calcium were evoked at EC(50) values of 59+/-4 and 37+/-7 microM, respectively. Both calcium release and inositol 1,4,5-trisphosphate (IP(3)) production induced by neurotensin suggested involvement of phospholipase C. Experiments with simultaneous or sequential treatment with neurotensin and bradykinin suggested that neurotensin and bradykinin act on the same binding sites. Furthermore, both inhibition of bradykinin- and neurotensin-induced calcium rises by bradykinin receptor antagonists with similar IC(50) values and receptor binding analysis using [3H]bradykinin confirmed that neurotensin directly binds to B2 bradykinin receptors. The data suggest that neurotensin binds and activates the B2 bradykinin receptors.     

Ontogeny and distribution of certain endocrine cells in the human fetal large intestine

Summary In 9 fetuses, 9 to 24 weeks-old, the occurrence and relative distribution of argentaffin cells, as well as of cells immunoreactive to somatostatin (SRIF), glucagon-like polypeptide (GLI), pancreatic polypeptide (PP) and substance P (SP) were studied in five segments of the colon (appendix, cecum, ascending colon, descending colon, and rectosigmoid). For each colonic segment, data concerned with the occurrence of endocrine cells were expressed either as mean absolute numbers of specific cells per entire mucosal section, or as cell densities per mm³ of mucosa after calculation of the mucosal volume of the sections. Argentaffin, GLI, SRIF and PP immunoreactive cells are all present in relatively large numbers, scattered along the entire length of the colonic mucosa as early as the 9th–10th week of gestation, whereas substance P-containing cells occur sporadically and first appear during the 14th–17th week. Until the 20th week, with progressing embryonic development, an increase was determined in absolute numbers per section of all types of endocrine cells in all segments of the colon. This observation is clearly related to the general growth of the colonic mucosa, since cell densities per mm³ of mucosa do not greatly change or even decrease during gestation. However, it is possible that densities of argentaffin, GLI and BPP cells increase in the appendix around the 14th–17th week of gestation. Between the 20th and 24th week, absolute numbers of cells per section remain stable or slightly increase, while cell densities tend rather to decrease in all segments. These data demonstrate that some endocrine cells are present very early in the human fetal colon, but their functional significance remains to be elucidated.This work was supported by the Institut National de la Santé et de la Recherche Médicale (INSERM)     

Anomeric specificity in the response of gut glucagon-like immunoreactive materials to glucose.

An anomeric specificity of the glucose sensors of A cells and B cells of the pancreas has been reported. In this context the present authors investigated, using the canine intestinal loop prepared from the terminal portion of the ileum, how glucagon-like immunoreactive materials (GLI) of the gut would respond to glucose anomers in an attempt to explore a possible anomeric specificity of glucose-stimulated gut GLI secretion. As a result GLI was found to be more readily released into the blood stream after an intestinal alpha-glucose load than following beta-gluocse during a 15-minute observation period. It is thus suggested that gut GLI-secreting cells have glucose sensors similar to those of pancreatic A or B cells which are specific for the alpha-glucose anomer.     

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.     

A Novel Zinc Finger Protein, Zic, Is Involved in Neurogenesis, Especially in the Cell Lineage of Cerebellar Granule Cells

Abstract: To clarify the mechanism of cerebellar development, we have cloned a gene, named zic, encoding a zinc finger protein that is expressed abundantly in granule cells throughout development of the cerebellum. zic has a significant homology to the zinc finger domain of the Caenorhabditis elegans tra1 gene, the Drosophila cubitus interruptus Dominant gene, and the human GLI oncogene. An in situ hybridization study revealed that zic showed a restricted expression pattern in the granule cells and their putative precursor cells. It is also expressed at an early embryonic stage in the dorsal half of the neural tube. The expression pattern and nuclear localization were confirmed by immunohistochemical study. Furthermore, the bacterially expressed zic protein containing the zinc finger domains bound to the GLI -binding sequence. These findings suggest that zic is one of a number of nuclear factors involved in both differentiation in early development and maintenance of properties of the cerebellar granule cells.     

大鼠纹状体边缘区内神经降压肽和生长抑素免疫阳性反应的分布

用免疫组织化学方法研究了大鼠纹状体边缘区中神经降压肽和生长抑素免疫阳性反应的分布。神经降压肽和生长抑素免疫阳性纤维和胞体散在分布于纹状体中,但在边缘区中分布密集,形成一条明显的带,带的宽度和位置和边缘区一致。边缘区中可见神经降压肽和生长抑素免疫阳性胞体。本研究证明纹状体边缘区中存在密度较高的神经降压肽和生长抑素免疫阳性纤维和胞体,并推测和边缘区的学习记忆功能有关     

Immunohistochemical investigations of neuropeptides in the brain,corpora cardiaca,and corpora allata of an adult lepidopteran insect,Manduca sexta (L)

In the brain of adult specimens of the tobacco hornworm moth, Manduca sexta (L), cells immunoreactive for several kinds of neuropeptides were localized by means of the PAP procedure, by use of antisera raised against mammalian hormones or hormonal peptides. In contrast, no such neurosecretory cells were found in the corpora cardiaca and corpora allata (CC/CA); in the CC/CA, however, immunoreactive nerve fibres were observed, reaching these organs from the brain. The neurosecretory cells found in the brain were immunoreactive with at least one of the following mammalian antisera, namely those raised against the insulin B-chain, somatostatin, glucagon C-terminal, glucagon N-terminal, pancreatic polypeptide (PP), secretin, vasoactive intestinal polypeptide (VIP), glucose-dependent insulinotropic peptide (GIP), gastrin C-terminus, enkephalin, alpha- and beta-endorphin, Substance P, and calcitonin. No cells were immunoreactive with antisera specific for detecting neurons containing the insulin A-chain, nerve growth factor, epidermal growth factor, insulin connecting peptide (C-peptide), polypeptide YY (PYY), gastrin mid-portion (sequence 6-13), cholecystokinin (CCK) mid-portion (sequences 9-20 and 9-25), neurotensin C-terminus, bombesin, motilin, ACTH, or serotonin. All the neuropeptide-immunoreactive cells observed emitted nerve fibers passing through the brain to the CC and in some cases also to the CA. In CC these immunoreactive nerve fibers tended to accumulate near the aorta. It was speculated that neuropeptides are released into the circulating haemolymph and act as neurohormones.     

Ultrastructure of the gut neurotensin cell

Summary In mammals, neurotensin cells occur scattered in the epithelium of the jejunum-ileum. In chicken, neurotensin cells are abundant in the region of the gizzard-duodenal junction (antrum) where they occur intermingled with numerous somatostatin and gastrin cells. The neurotensin cells in chicken, dog and man were identified at the electron microscopic level by immunocytochemistry, using the consecutive semithin/ultrathin section technique. They contain numerous electron dense cytoplasmic granules, predominantly in the basal portion of the cell. It was shown that these granules are the storage site for neurotensin. The neurotensin granules are round, highly electron dense and of about the same size in the different species examined (mean diameter 260–290 nm). in dog and man the granules have a tightly applied surrounding membrane while in the chicken a relatively electron lucent zone separates the electron dense core from the granule membrane. The ultrastructure of the neurotensin granules in chicken is some-what reminiscent of that of the gastrin granules. The mean diameter of the gastrin granules in chicken antrum is 230 nm; for the somatostatin granules the mean diameter is 305 nm.     

Ultrastructure of the gut neurotensin cell.

In mammals, neurotensin cells occur scattered in the epithelium of the jejunum-ileum. In chicken, neurotensin cells are abundant in the region of the gizzard-duodenal junction (antrum) where they occur intermingled with numerous somatostatin and gastrin cells. The neurotensin cells in chicken, dog and man were identified at the electron microscopic level by immunocytochemistry, using the consecutive semithin/ultrathin section technique. They contain numerous electron dense cytoplasmic granules, pre-dominantly in the basal portion of the cell. It was shown that these granules are the storage site for neurotensin. The neurotensin granules are round, highly electron dense and of about the same size in the different species examined (mean diameter 260--290 nm). In dog and man the granules have a tightly applied surrounding membrane while in the chicken a relatively electron lucent zone separates the electron dense core from the granule membrane. The ultrastructure of the neurotensin granules in chicken is somewhat reminiscent of that of the gastrin granules. The mean diameter of the gastrin granules in chicken antrum is 230 nm; for the somatostatin granules the mean diameter is 305 nm.     

Ontogeny of glucagon-like immunoreactive peptides in rat intestine

The ontogeny of the intestinal glucagon-like peptides was investigated in rats between 16 days of gestation and 4 postnatal days. The intestinal content of glucagon-like immunoreactive (GLI) peptides increased from 0.09 +/- 0.02 pmol/nmol protein at 16-17 days to plateau at 2.8 +/- 0.4 pmol/nmol protein by 20 days of gestation (P less than 0.001). The apparent immunoreactive glucagon (IRGa) content of the gut ranged from 0.03 +/- 0.01 to 0.08 +/- 0.01 pmol/nmol protein. No developmental trends in IRGa peptide content were observed. Following gel filtration of intestines extracted from rats of 18 days of gestation or greater, two main GLI peptides were detected with apparent mol. wts. of 11-12 and 5-6 kDa. Significant peaks of GLI peptides were not detected following gel filtration of intestines extracted from 16- or 17-day fetuses, nor were peaks of IRGa found at any age. In conclusion, the fetal rat intestine undergoes maturational development between 17 and 19 days of gestation to produce the GLI peptides.     

Occurrence of neurotensin-immunoreactive cells in the digestive tract of lower vertebrates and deuterostomian invertebrates

Summary In the mucosal epithelium of the digestive tract of two marine teleost bony fish, one cartilaginous fish, one cyclostome, and in that of two of three representatives of deuterostomian invertebrates studied, endocrine cells of open type were found, exhibiting immunoreactivity with antisera against C-terminal sequences of mammalian neurotensin and of the structurally closely related amphibian neurohormonal peptide xenopsin.From these observations, and from those of previous studies, it is suggested that neurotensin cells do not occur in the digestive tract mucosa until at the evolutionary level of the more highly developed deuterostomian invertebrates. Three evolutionary stages seem to exist in the distribution pattern. The first stage, characterized by few, widely scattered cells, is found in the uro- and cephalochordates, the cyclostomes, the cartilaginous fish, and the stomachless bony fish. In the second stage, comprising the remaining submammalian classes, including more highly developed bony fish, the typical distribution pattern is that of numerous neurotensin immunoreactive cells in the antrum, pylorus, and duodenum. The final stage of neurotensin evolution is found in higher mammals and is characterized by a great density of neurotensin immunoreactive cells in the ileum.Dedicated to Prof. Dr. J. Staubesand on the occasion of this 60th birthday     

Regulation of cyclic GMP levels by neurotensin in neuroblastoma clone N1E115

The binding of 125I-labeled [monoiodo-Tyr3]neurotensin to intact neuroblastoma N1E115 cells and the effect of neurotensin on the intracellular concentration of cyclic nucleotides were studied at 37 degrees C and under physiological conditions of pH and ionic strength. The radiolabeled neurotensin analogue bound specifically to differentiated cells with a dissociation constant of 0.75 nM and a maximal binding capacity of 45 fmol/10(6) cells. Incubation of neuroblastoma cells with neurotensin in the presence of calcium ions resulted in a transient increase of 10 fold over basal level of the intracellular cyclic GMP concentration. Half-maximal stimulation was obtained with 2 nM neurotensin. Under identical conditions the cyclic AMP concentration only decreased by 20-30%. These results suggest that cyclic GMP is a second messenger of neurotensin in neuroblastoma clone N1E115.