The Regional Distribution and Chromatographic Characterisation of Neurotensin-Like Immunoreactivity in the Rat Central Nervous System

Abstract: Carboxy- and amino-terminal specific neurotensin antisera have been characterized and used to determine the nature of neurotensin-like immunoreactivity in the rat central nervous system. Using these antisera, together with the separation of neurotensin-like immunoreactivity on reversephase HPLC columns, it is clear that the majority of rat central nervous system neurotensin-like immunoreactivity is indistinguishable from the synthetic tridecapeptide. However, smaller amounts of carboxy- and amino-terminal neurotensin-like immunoreactivity were detected, which may correspond to carboxy- and amino-terminal fragments of neurotensin. In addition, using the amino-terminal directed neurotensin antiserum, a detailed distribution of neurotensin-like immunoreactivity in the rat central nervous system is described. Highest amounts were found in the hypothalamus, central amygdaloid nucleus, bed nucleus of the stria terminalis and the substantia gelatinosa of the spinal cord and of the trigeminal region.     

Distribution of neurotensin-like immunoreactivity in the central nervous system of the Formosan monkey

The distribution of neurotensin-like immunoreactivity was investigated in the central nervous system of the Formosan monkey employing immunohistochemical techniques. Neurotensin-containing cells were found to be widely distributed in the forebrain. The principal densities of neurotensin-like neuronal perikarya were located in the limbic system, the basal ganglion and the cerebral cortex; particularly in the amygdala, the septum, the neostriatum, the claustrum and the insula. The stria terminalis and the preoptic area were also rich in immunostained neurotensin-like neurons. A large number of immunoreactive fibers were observed from the cerebral cortex to the spinal cord in locations such as the median eminence, the arcuate nucleus, the hippocampus, the central gray and the dorsal horn of the spinal cord. We analyzed in detail the distribution of neurotensin-like immunoreactivity in the brain of the Formosan monkey, and compared these results with those obtained in the brain of the rat, Japanese monkey and human. Some possible implications regarding differences in location of this peptide are also briefly discussed.     

Coexistence of substance P and neurotensin-like peptides in single neurons of the rat hypothalamus

The coexistence of substance P with neurotensin-like immunoreactivity in certain neurons of the hypothalamus were demonstrated by the double immunofluorescence method. Substance P and neurotensin-like immunoreactivity coexisted within single neurons of some hypothalamic areas such as the medial preoptic area, perifornical area, anterior hypothalamic area, lateral hypothalamic area, periventricular nucleus and posterior hypothalamic nucleus, although they did not coexist in the majority of immunoreactive cells.     

Transient appearance of substance P-like immunoreactivity in the granular convoluted tubules of the male mouse submandibular gland: light- and electron-microscopic studies

The time of appearance and distribution of substance P (SP)-like immunoreactivity in the granular convoluted tubule cells of the developing male mouse submandibular glands were examined, and the subcellular localization of SP-like immunoreactivity was investiagted by electron microscopy. At 25 days of age, SP-like immunoreactivity was first detected in the supranuclear cytoplasm of the granular convoluted tubule cells, which occurred either singly or in small clusters. At 30 and 35 days of age, granular convoluted tubule cells with SP-like immunoreactivity were more numerous than in the earlier stages, as the volume ratio of the cells increased. Not all granular convoluted tubule cells demonstrated SP-like immunoreactivity. The number of cells with SP-like immunoreactivity decreased at 60 days of age, and these cells had completely disappeared at 90 days of age. Most, but not all, secretory granules in the granular convoluted tubule cells were strongly labeled with gold particles, indicating that the subcellular site of SP-like substance is in the secretory granules within the cells. The findings suggest that the physiological role of the SP-like substance secreted from the GCT cells is restricted to the early postnatal stages, and that it may be involved in the development of the oral mucosa or digestive tract as a trophic factor.     

Neurotensin-like immunoreactivity generated by pepsin from human plasma and gastric tissue

The present study demonstrates precursors of neurotensin-like immunoreactivity (NTLI) endogenous to human gastric tissue and plasma, and the existence of a gastric NTLI-generating enzyme system. The molecular size of the NTLI-precursors in plasma and gastric tissue were estimated by gel permeation chromatography to be ca 50,000-60,000 and 60,000-70,000 Da, respectively. The neurotensin-like peptide generated from the precursor was detected with a carboxyl-terminally directed antiserum but did not cross-react with an amino-terminally directed antiserum. A neurotensin-like peptide isolated from pepsin-treated human plasma was characterized by mass spectrometry and its amino acid sequence determined. This novel nonapeptide, referred to as kinetensin, failed to affect pentagastrin-stimulated acid secretion or blood pressure in the rat. Sequence homologies between neurotensin, kinetensin and proteins of the serum albumin family suggest a common evolutionary origin and raise questions regarding albumin-like proteins as precursors of regulatory peptides.     

Isolation and characterization of a neurotensin-like decapeptide from a canine upper small intestinal extract

Neurotensin-like immunoreactivity can be detected in extracts of canine upper gastrointestinal mucosa when measured by carboxyl terminal but not by amino terminal antibodies to neurotensin. The nature of this immunoreactive material was characterized by complete purification on gel filtration and HPLC followed by peptide microsequence analysis. The structure obtained was Glu-Asn-Lys-Pro-Arg-Arg-Pro-Tyr-Ile-(Leu), identical in structure to the carboxyl terminal decapeptide of neurotensin. It cannot, however, be excluded that this neurotensin decapeptide was generated from a larger neurotensin-like peptide during the extraction procedure by a physiological or artificial enzymatic process. Since carboxyl terminal neurotensin fragments containing eight or more residues have full biological activity, this peptide may be responsible for neurotensin-like biological activities within the mucosa of, or after release from, the upper gut.     

The amino acid sequence of kinetensin, a novel peptide isolated from pepsin-treated human plasma: homology with human serum albumin, neurotensin and angiotensin

A novel nonapeptide with neurotensin-like immunoreactivity was isolated from pepsin-treated human plasma by dialysis, ion-exchange chromatography and high performance reversed-phase liquid chromatography. The amino acid sequence was determined by automated gas-phase sequence analysis as Ile-Ala-Arg-Arg-His-Pro-Tyr-Phe-Leu. Sequence homology with human serum albumin and with the biologically active peptides neurotensin and angiotensin is demonstrated. The name proposed for this peptide is kinetensin.     

Plasma neurotensin release and gastric emptying in the dumping syndrome

Seventy-three patients were studied after ingesting a liquid glucose meal, tagged with ¹¹³Indium. Nineteen of these patients were awaiting surgery for their duodenal ulcer, while 54 were studied postoperatively, 25 of whom experienced troublesome postprandial (dumping) symptoms in their daily lives. The radioactive marker emptied significantly faster in the symptomatic patients than in the symptomfree, pre and post-operative groups (initial emptying rate 3.45 ± 0.23, compared with 1.16 ± 0.19 and 1.27 ± 0.15% fall in counts/min respectively; p < 0.01). Initial (20 min) rises in the plasma concentrations of neurotensin-like immunoreactivity measured during the test correlated significantly with the rate of gastric emptying in all patients, being greatest in patients with dumping symptoms. Physiological concentrations of neurotensin have been shown to delay gastric emptying. The excessive rise in plasma neurotensin-like immunoreactivity in patients with dumping symptoms, presumably occuring as a result of the rapid passage of nutrients to the neurotensin-rich ileum, may possibly have a compensatory role in slowing further emptying from the stomach.     

Comparison of Changes in GAD65 and GAD67 Immunoreactivity and Levels in the Gerbil Main Olfactory Bulb Induced by Transient Ischemia

In the present study, we investigated changes in glutamate decarboxylase 65 (GAD65) and GAD67 immunoreactivity and protein levels in the main olfactory bulb (MOB) after 5 min of transient forebrain ischemia in gerbils. GAD65 immunoreactivity in the sham-operated group was shown in neurons and neuropil except for the somata of granule cells. GAD65 immunoreactivity was increased in neurons in the external plexiform layer 60 days after ischemia, and in mitral cells 30 and 60 days after ischemia. GAD67 immunoreactivity in the sham-operated group was shown in periglomerular cells, neuron in the external plexiform layer and granule cells with neuropil. GAD67 immunoreactivity in periglomerular cells was increased 10, 45 and 60 days after ischemia. GAD67 immunoreactivity in neurons in the external plexiform layer was increased 10 and 15 days after ischemia. Mitral cells showed strong GAD67 immunoreactivity 10 days after ischemia. However, GAD67 immunoreactivity in the granule cells was not changed with time after ischemia. In Western blot analysis for GAD65 and GAD67 protein levels in the ischemic gerbil MOB, GAD65 level was not changed after ischemia; GAD67 level was increased 10 days after ischemia. These results suggest that transient ischemia causes changes in GAD65 and GAD67 immunoreactivity in the gerbil MOB, and this change may induce a malfunction in olfaction after an ischemic insult. Ki-Yeon Yoo and In Koo Hwang equally contributed to this article.     

Ultrastructural immunocytochemical studies on CCK-and neurotensin-like immunoreactivity in the nucleus accumbens of the rat

Using the peroxidase-antiperoxidase technique of Sternberger and collaborators the ultrastructural localization of cholecystokinin- and neurotensin-like immunoreactivity was studied in the nucleus accumbens of the albino rat. The immunoreactive material was localized to nerve terminals making probable axodendritic or axo-axonic contacts with neighbouring nerve cells. Within the nerve terminals most immuno-reactivity was confined to so-called large dense-core vesicles.     

Characterization of neurotensin(6-13) from an hepatic fibrolamellar carcinoma.

Neurotensin(6-13) has been isolated and sequenced as the major form of neurotensin-like immunoreactivity (NTLI) in a human hepatic fibrolamellar carcinoma. Circulating NTLI in the patient, especially C-terminal, was very high. In additional studies, NT(6-13) was synthesized and compared with the purified tumor NTLI by HPLC analysis and by testing stability in plasma in vitro. These methods confirmed that the tumor NTLI was identical to NT(6-13). Since the metabolic clearance rate of synthetic NT(6-13) in sheep was 30-fold higher than NT(1-13), it suggests that the elevated plasma levels are the result of impaired clearance and/or markedly elevated production.     

Changes in Leydig cell function during sexual maturation in the mouse

Changes in androgen production by isolated Leydig cells were evaluated from 20 through 60 days of age in the mouse. Leydig cells were obtained by mechanical dissociation of testes, purified by centrifugation in metrizamide gradients, and incubated with increasing concentrations of human chorionic gonadotropin (hCG). Testosterone and 5 alpha-androstane-3 alpha, 17 beta-diol (androstanediol) were measured by radioimmunoassay in samples of cells plus medium. Sensitivity of mouse Leydig cells, evaluated as the concentration of hCG that elicited half-maximum androgen responses, was essentially the same at all ages. Maximum testosterone production increased by about 20-fold from 20 to 45 days of age but was no greater at 60 days than at 45 days. Maximum androstanediol production increased by about 3- to 4-fold from 20 to 25 days and declined after 30 days of age. Androstanediol predominated over testosterone by about 2-fold at 20 days; this relationship was reversed by 30 days, and at later ages testosterone greatly predominated over androstanediol (by at least 4- and 6-fold at 45 and 60 days of age, respectively). Maximum total androgen production, estimated from the sum of the values for testosterone and androstanediol, increased by about 7-fold from 20 to 30 days of age and remained essentially constant thereafter. These results are compared with those from previous studies of the rat.     

VIP-, Bombesin- and Neurotensin-Like Immunoreactivity in Neurons of the Gut of the Holostean Fish,Lepisosteus platyrhincus

VIP-like immunoreactivity was found in nerve fibres in all layers of the gut wall in both stomach and intestine, and was abundant in the myenteric and submucous plexuses. A few fibres were associated with blood vessels. Nerve cells showing VIP-like immunoreactivity were found in the myenteric plexus. Neurotensin-like immunoreactivity was found in nerve cells and numerous nerve fibres in the myenteric plexus of both stomach and intestine and in nerve fibres of the circular muscle layer, while bombesin-like immunoreactivity was confined to a low number of nerve fibres in the myenteric plexus of the stomach. The results indicate that a VIP-like, a neurotensin-like and a bombesin-like peptide are present in neurons of the gut of Lepisosteus.     

Effect of Dietary Vanadium on the Ileac T Cells and Contents of Cytokines in Broilers

The purpose of this 42-day study was to examine the effect of dietary vanadium on the ileac T cells and contents of cytokines including interleukin-2 (IL-2), interleukin-6 (IL-6), and interferon-gamma (IFN-γ) in broilers by flow cytometry and enzyme-linked immunosorbent assay. A total of 420 one-day-old avian broilers were divided into six groups (seven replicates in each group and ten broilers in each replicate) and fed on control diet or the same diet supplemented with 5, 15, 30, 45, and 60 mg/kg vanadium in the form of ammonium metavanadate. The results showed that the percentages of CD3(+), CD3(+)CD4(+), and CD3(+)CD8(+) T cells in both ileac lamina propria lymphocytes (LPLs) and intraepithelial lymphocytes (IELs) were significantly lower (P < 0.05 or P < 0.01) in the 45- and 60-mg/kg groups than in the control group from 14 to 42 days of age. The CD4(+)/CD8(+) ratio was increased in ileac LPLs in the 60-mg/kg group at 28 days of age, and in ileac IELs in the 60-mg/kg group at 28 days of age and in the 45-mg/kg group at 42 days of age. Meanwhile, the ileac IL-2, IL-6 contents were decreased (P < 0.05 or P < 0.01) in the 60-mg/kg group from 14 to 42 days of age and in the 45-mg/kg group from 28 to 42 days of age in comparison with those of the control group. It was concluded that dietary vanadium in excess of 30 mg/kg reduced the ileac T cell population and percentages of T cell subsets, and IL-2, IL-6, and IFN-γ contents, implying that the immune function of local intestinal mucosa in broilers could be affected by the dietary vanadium.     

Tachykinins (Substance P, Neurokinin A and Neuropeptide γ) and Neurotensin from the Intestine of the Burmese Python, Python molurus

Conlon, J. M., T. E. Adrian and S. M. Secor. Tachykinins (substance P, neurokinin A and neuropeptide γ,) and neurotensin from the intestine of the burmese python, Python molurus. Peptides 18(10) 1505–1510, 1997.—Peptides with substance P-like immunoreactivity, neurokinin A-like immunoreactivity and neurotensin-like immunoreactivity were isolated in pure form from an extract of the intestine of the Burmese python (Python molurus). The primary structure of python substance P (Arg-Pro-Arg-Pro-Gln-Gln-Phe-Tyr-Gly-Leu-Met-NH₂) shows one amino acid substitution (Phe⁸ → Tyr) compared with chicken/alligator substance P and an additional substitution (Lys³ → Arg) as compared with mammalian substance P. The neurokinin A-like immunoreactivity was separated into two components. Python neuropeptide γ (Asp-Ala-Gly-Tyr-Ser-Pro-Leu-Ser-His-Lys-Arg-His-Lys-Thr-Asp-Ser-Phe-Val-Gly-Leu-Met-NH₂ shows three substitutions (Gly⁵ → Ser, Gln⁶ → Pro and Ile⁷ → Leu) compared with alligator neuropeptide γ and an additional substitution (His⁴ → Tyr) compared with mammalian neuropeptide γ. Python neurokinin A (His-Lys-Thr-Asp-Ser-Phe-Val-Gly-Leu-Met.NH₂) is identical to human/chicken/alligator neurokinin A. Python neurotensin (pGlu-Leu-Val-His-Asn-Lys-Ala-Arg-Pro-Tyr-Ile-Leu) is identical to chicken/alligator neurotensin. The data are indicative of differential evolutionary pressure to conserve the amino acid sequences of reptilian gastrointestinal peptides.     

猪脂肪组织中IGF2和IGFBP3基因表达的发育性变化及其品种差异

采用荧光定量PCR技术分析长白猪和太湖猪背部皮下脂肪组织中胰岛素样生长因子2和胰岛素样生长因子结合蛋白3基因在30、60、90、120和150日龄时表达水平的发育性变化。结果表明: (1)品种内日龄间比较, 长白猪IGF2 mRNA 在30日龄时的表达量极显著高于其他日龄(P<0.01), 之后逐渐下降, 至120日龄降到最低, 150日龄时又明显上升; 太湖猪IGF2 mRNA在30~60日龄的表达量较高, 90日龄降至最低, 120日龄迅速回升, 之后又有所下降。两品种IGFBP3 mRNA表达的发育性变化模式基本相同, 30日龄时表达量最高, 60日龄显著下降(P<0.05), 之后趋于平缓但略有波动。(2)品种间同日龄比较, 120日龄时太湖猪IGF2 mRNA的表达量极显著高于长白猪(P<0.01), 150日龄时太湖猪IGFBP3的表达量极显著低于长白猪(P<0.01), 其余日龄间两品种差异均不显著(P>0.05)。研究结果提示: 猪脂肪组织IGF2和IGFBP3基因表达存在明显的发育性变化和品种差异; IGF2基因表达水平的降低可能与脂肪细胞的增殖有关。     

Induction of secretory phospholipase A2 in reactive astrocytes in response to transient focal cerebral ischemia in the rat brain

Although mRNA expression of group IIA secretory phospholipase A2 (sPLA2-IIA) has been implicated in responses to injury in the CNS, information on protein expression remains unclear. In this study, we investigated temporal and spatial expression of sPLA2-IIA mRNA and immunoreactivity in transient focal cerebral ischemia induced in rats by occlusion of the middle cerebral artery. Northern blot analysis showed a biphasic increase in sPLA2-IIA mRNA expression following 60-min of ischemia-reperfusion: an early phase at 30 min and a second increase at a late phase ranging from 12 h to 14 days. In situ hybridization localized the early-phase increase in sPLA2-IIA mRNA to the affected ischemic cortex and the late-phase increase to the penumbral area. Besides sPLA2-IIA mRNA, glial fibrillary acidic protein (GFAP) and cyclo-oxygenase-2 mRNAs, but not cytosolic PLA2, also showed an increase in the penumbral area at 3 days after ischemia-reperfusion. Immunohistochemistry of sPLA2-IIA indicated positive cells in the penumbral area similar to the GFAP-positive astrocytes but different from the isolectin B4-positive microglial cells. Confocal microscopy further confirmed immunoreactivity of sPLA2-IIA in reactive astrocytes but not in microglial cells. Taken together, these results demonstrate for the first time an up-regulation of the inflammatory sPLA2-IIA in reactive astrocytes in response to cerebral ischemia-reperfusion.     

Neurotensin interacts with carbachol, secretin, and caerulein in the stimulation of the exocrine pancreas of the rat in vitro

In the present investigation the effect of neurotensin on pancreatic secretion of isolated pancreatic lobules from the rat was examined. We found a dose- and time-dependent stimulation of amylase release beginning with a concentration of 10(-9) M neurotensin. This response was potentiated by the cholinergic agonist carbachol, the gastrointestinal peptide secretin, and the CCK analogue caerulein. As we found neurotensin-immunoreactive nerves within the pancreas and as neurotensin-like immunoreactivity is present in the circulation (found previously), neurotensin may well be a further peptide taking part in the regulation of exocrine pancreatic secretion either as a hormone or a neurotransmitter. Neurotensin would then cooperate with cholinergic mechanisms, secretin, and CCK.     

Effects of environment on morphology of rat cerebral cortex and hippocampus.

Cortical depth differences were found between male rats exposed to enriched or impoverished environmental conditions for successively shorter times, i.e., for 15 days (from 25 to 40 days of age), for seven days (25 to 32 days of age), and for four days (26 to 30 or 60 to 64 days of age). If the experiments began at weaning (at 25 days of age), the cortical depth changes were caused primarily by impoverishment. If the animals were young adults (60 days of age), upon entering their respective conditions, the cortical changes were induced by enrichment. No cortical depth differences were found between enriched and impoverished rats after one day of differential experience, from 60 to 61 days of age. In every age group and for every duration, except for the one day group, the dorsal-medial segment of the occipital cortex responded to the environmental conditions. No significant hippocampal depth differences were noted between enriched and impoverished animals.     

Performance of Holstein calves receiving equal quantities of milk at fixed or variable amounts per day during milk-feeding period

Two experiments were conducted to determine the effects of feeding equal quantities of milk during the pre-weaning period through different milk-feeding regimes on calf growth, starter intake and selected blood metabolites. In experiment 1, 44 female Holstein calves (3 days of age and 39.2±4.3 kg of BW) were distributed randomly to one of two milk-feeding programs (1 calf per pen; 22 pens per treatment group): (1) consistent (CONS; 6 l/day of milk from days 3 to 60 and 3 l/day from days 61 to 65 of age) or (2) step-up/step-down (SUSD; 5 l/day of milk from days 3 to 15, 8 l/day from days 16 to 40, 6 l/day from days 41 to 50, 3 l/day from days 51 to 60 and then 2 l/day from days 61 to 65 of age). No difference between treatments was observed in starter consumption, feed efficiency, hip width and heart girth. However, pre-weaning average daily gain (ADG) tended to be greater in CONS than in SUSD calves (0.78 v. 0.70 kg/day; P=0.07). Blood β-hydroxybutyrate at day 45 (pre-weaning) was lower in SUSD than in CONS calves (0.14 v. 0.21±0.013 mmol/l). In experiment 2, 26 male Holstein calves (3 days of age and 39.4±4.1 kg of BW) were assigned at random to one of two milk-feeding protocols (1 calf per pen; 13 pens per treatment group): (1) consistent (CONS; (7 l/day of milk from days 3 to 40 and 2 l/day from days 41 to 45 of age) or (2) step-down (STD; 8 l/day of milk from days 3 to 30, 4 l/day from days 31 to 40 and 2 l/day from days 41 to 45 of age). The milk-feeding program had no effect on the performance measurements, with the exception that ADG (days 15 to 30), starter intake (days 30 to 45) and heart girth (day 45) were greater in STD than in CONS calves. In conclusion, it appears that if the total amount of milk intake is held constant over the course of milk-feeding period, the method of milk feeding would have negligible effects on calf performance.