Release Of Neurotensin-like Immunoreactivity Research Articles

Functional coupling between the active transport of glucose and the secretion of intestinal neurotensin in rats.

1. In this study, the mechanisms involved in the release of neurotensin-like immunoreactivity (NTLI) by glucose were investigated with the isolated, vascularly perfused rat jejunoileum preparation. 2. Luminal infusion of glucose (1-250 mM) produced a sharp and sustained release of NTLI in the intestinal venous effluent. The first significant response was observed with 5 mM glucose and the release reached a maximum under 250 mM glucose with a plateau secretion at 500% of basal. 3. There was no significant difference in the ability of galactose and 3-O-methylglucose to release NTLI when compared to glucose, but alpha-methylglucose, mannose, 2-deoxyglucose and fructose did not stimulate NTLI release. 4. Luminal infusion of 5 mM phloridzin reduced the glucose-induced release of NTLI by 90%. Intra-arterial infusion of glucose (25 mM) or of phloretin (20 microM) had no significant effect on the glucose-evoked NTLI secretion. 5. Intra-arterial infusion of ouabain (1 mM) produced a dramatic increase (at about 1500% of basal) in portal NTLI although it drastically reduced intestinal absorption of glucose. 6. Intra-arterial infusion of tetrodotoxin (1 microM), atropine (10 microM), verapamil (50 microM) or nifedipine (50 microM) did not modify the glucose-induced NTLI secretion. 7. Intra-arterial infusion of forskolin (2-20 microM) evoked a prompt and well-sustained secretion of NTLI which was increased to a mean value of 800% of basal with the highest dose tested. 3-Isobutyl-1-methylxanthine (IBMX, 10-100 microM) also stimulated the secretion of NTLI (maximal increase at 725% of basal at 100 microM). In contrast, intra-arterial infusion of 4-beta-phorbol 12-myristate, 13-acetate (PMA, 0.05-0.5 microM) had no effect on NTLI release. 8. IBMX (10-100 microM) synergistically enhanced NTLI responses induced by 250 mM glucose; the integrated response of NTLI release was 3- to 5-fold higher than the sum of individual responses produced by the same stimulants given separately. 9. It is concluded that the carbohydrate-induced NTLI release is related to the active, sodium-dependent hexose transport, but not to the carbohydrate catabolic pathway. Furthermore, the intramural nerves and L-type calcium channels are not involved in the glucose-induced NTLI secretion. Finally, the secretory activity of the intestinal N cell seems to be mainly stimulated through a cAMP-dependent pathway.

Fatty acids stereospecifically stimulate neurotensin release and increase [Ca2+]i in enteric endocrine cells

In primary cultures of canine enteric endocrine cells, fatty acids directly stimulated the release of neurotensin-like immunoreactivity (NTLI). This stimulatory effect was cell specific, selective for long-chain unsaturated fatty acids, and stereospecific. Saturated fatty acids of comparable chain length and trans isomers of long-chain unsaturated fatty acids had no effect on basal NTLI secretion. NTLI release in response to oleic acid (cis-11) was dose dependent with an apparent EC50 of 37 +/- 0.18 microM. Cyclooxygenase inhibitors had no effect on fatty acid-stimulated NTLI release, indicating the response was not mediated by the production of active arachidonic acid metabolites. Somatostatin (100 nM) inhibited maximal oleic acid-stimulated NTLI release by 92%. Long-chain unsaturated fatty acids also selectively and stereospecifically stimulated an increase in the mobilization of [Ca2+]i to 313.5 +/- 28.6% of resting [Ca2+]i. Staurosporine, an inhibitor of protein kinase C, dose dependently inhibited oleic acid-stimulated NTLI release with an IC50 value of 22 +/- 0.4 nM. Long-chain unsaturated fatty acids had no effect on basal NTLI secretion from rat pheochromocytoma cells and medullary thyroid carcinoma cells, two clonal lines that express NTLI. The cell-specific, selective stereospecific, and inhibitable action of fatty acids on NTLI secretion suggests that the effect of fatty acids on enteric endocrine cells is indicative of a receptor-mediated mechanism.

The localization of the site(s) of neurotensin release in man and dog

AbstractThe major source of neurotensin in the gut is the ‘N’ cell and this is found in the highest density in the ileum. The ingestion of food, particularly fat, causes the biphasic release of neurotensin‐like immunoreactivity (NTLI) into the circulation. The early peak occurs sooner than expected if it is due to the presence of chyme in the ileum, suggesting that the early release of neurotensin is due to a more proximal concentration of N cells or that neurotensin is released from the ileum by a more proximal stimulus. This paper investigates the site(s) of neurotensin release by studying: three groups of patients with various resections of the small intestine and the fashioning of duodenostomies, jejunostomies and ileostomies; and the dog with chronic gastric, duodenal and ileal fistulae, and following ileal resection. The results indicate that the jejunum and ileum are critical in the release of neurotensin following fat stimulation and that the stomach and duodenum have no direct role. The stimulation of the jejunum by fat causes the early rise of plasma NTLI by releasing neurotensin from the ileum. If chyme is prevented from passing to the distal jejunum the magnitude of the early peak is diminished and the second, later, peak is abolished. The second peak of plasma NTLI is due to the direct luminal stimulation of the distal jejunum and ileum by the products of fat digestion. Ileal resection completely abolishes any release of plasma NTLI in response to fat. It was concluded that the source of neurotensin released by the ingestion of food is the ileum. The release of neurotensin from the distal gut is dependent upon a signal from the proximal to the distal gut. The identity of the signal is unknown, but it is either neural or humoral and previous studies suggest a cholinergic‐dependent reflex.

Effect of renal failure or portacaval shunt on release of neurotensin in man.

Neurotensin-like immunoreactivity (NTLI) is released into the circulation following a meal. However, the factors influencing its degradation and metabolism are uncertain, but it is presumed that the kidneys and liver are involved. This study has determined the release of NTLI after a liquid fat meal (200 ml 20% Intralipid) in 10 healthy control subjects, five patients with stable chronic renal failure (CRF), and five patients after a portacaval shunt (PCS), to assess the possible role of the liver and kidney in NTLI metabolism. NTLI was measured by radioimmunoassay using both specific C and N terminally directed antisera. The results showed: Basal NTLI levels are similar in controls, after PCS, and in CRF. A liquid fat meal produces a single early peak of NTLI and levels are greater after PCS than in controls or CRF. Levels return much more slowly to basal in CRF and after PCS. Thus it is likely that both the liver and kidney are involved in the metabolism and clearance of NTLI.

Regulation of neurotensin release from canine enteric primary cell cultures.

A recently developed primary cell-culture system allows direct study of the cellular mechanisms regulating neurotensin secretion from intestinal mucosal cells. We now report the use of these methods to evaluate the modulation of neurotensin release by adrenergic, cholinergic, and peptidergic transmitters. Collagenase-dispersed canine ileal mucosal cells, enriched for neurotensinlike immunoreactivity (NTLI) by centrifugal elutriation, were maintained for 48 h on collagen-coated culture dishes. Epinephrine (0.01-100 microM) stimulated a dose-dependent increase increase in NTLI secretion. The NTLI response to epinephrine increase in NTLI secretion. The NTLI response to epinephrine was competitively inhibited by propranolol, producing a parallel rightward shift of the epinephrine dose-response curve. alpha-Adrenergic agonist methoxamine (10 microM) and clonidine (10 microM) did not alter basal NTLI secretion. Epinephrine stimulation was not significantly inhibited by the alpha-adrenergic antagonists prazosin (10 microM) or yohimbine (10 microM). The diterpene forskolin, an adenyl cyclase activator, increased NTLI release and had an additive effect on the response to epinephrine. In contrast to beta-adrenergic activation, carbachol and somatostatin produced a dose-dependent inhibition of epinephrine-stimulated NTLI release. At 100 microM carbachol, NTLI release was inhibited 68%, and this action was partially blocked by atropine (0.1 microM). Somatostatin (100 nM) produced a 96% inhibition that was not surmountable by 1 mM epinephrine. These data indicate that neurotensin release is stimulated by beta-adrenergic agonists and adenylate cyclase activation. Somatostatin and the muscarinic agonist carbachol directly inhibit NTLI release.

Isolated canine ileal mucosal cells in short-term culture: a model for study of neurotensin release.

We have developed a new technique for maintaining isolated enteric mucosal cells containing neurotensinlike immunoreactivity (NTLI) in short-term culture to determine the regulation of NTLI release. Collagenase-dispersed ileal mucosal cells, separated by centrifugal elutriation, were enriched for NTLI-containing cells as determined by immunohistochemistry and radioimmunoassay. Bombesin (BBS) rapidly stimulated NTLI release from freshly isolated cells. However, studies with freshly isolated cells were limited by high, unstable basal release measurements and rapid degradation of added [3H]neurotensin-(1-13). A culture system of elutriator-enriched NTLI cells was therefore developed. After 48 h NTLI cells selectively adhered to the collagen substrate and constituted 40% of the viable cells in culture. The morphology of NTLI cells in culture closely resembled neurotensin cells in intestinal tissue sections, with a secretory granule diameter of 292 +/- 14 nm. Bombesin stimulated a dose-dependent increase in NTLI secretion over 120 min. In these cell cultures, degradation of added [3H]-neurotensin-(1-13) was minimal over 120 min. High-pressure liquid chromatographic analysis of culture supernatants characterized neurotensin-(1-13) as the primary molecular form of neurotensin released in response to BBS stimulation. In conclusion, we have established a primary culture of enteric neurotensin cells that provides a model for studying the regulation of peptide release. Bombesin stimulation of NTLI release verified the functional responsiveness of this isolated cell system.

Effect of truncal vagotomy on neurotensin-like immunoreactivity release in dogs

To elucidate the relationship between the vagus nerve and circulating neurotensin release, mongrel dogs were given an intraduodenal infusion of a 50 ml water solution containing 10 g glucose (n = 4) or 5 g soybean oil (n = 7) over a period of 4 min before and after truncal vagotomy with pyloroplasty. In the prevagotomized animals only a slight increase of neurotensin-like immunoreactivity (NTLI) was observed following glucose infusion, while NTLI in response to fat infusion was significantly increased. After vagotomy, NTLI release following fat infusion was significantly decreased when compared to untreated control animals, suggesting that vagotomy causes a major alteration in circulating NTLI release and that the vagus nerve may play a definite role in fat-induced NTLI release.

The effect of atropine and vagal stimulation on the release of neurotensin-like immunoreactivity in man.

This study investigated the food stimulated release of neurotensin-like immunoreactivity (NTLI) in man with and without the administration of atropine, and the influence of vagal stimulation by modified sham feeding and insulin hypoglycaemia. NTLI was measured, after ethanol extraction, by specific C- and N-terminally directed antisera. With both a liquid fat meal and a mixed meal an early peak of NTLI occurred. The mixed meal also produced a second sustained rise in plasma NTLI. An intramuscular injection of 0.6 mg atropine sulphate abolished the early peak, but had no effect on the late peak. Modified sham feeding and insulin hypoglycaemia did not release NTLI. We conclude that it is possible that a cholinergic non-vagal mechanism is responsible for the early phase of food stimulated release of NTLI in man, and that the second sustained rise may be cholinergically independent.

Postprandial release of neurotensin-like immunoreactivity and its mechanism.

The effects of various ingested materials on plasma neurotensin-like immunoreactivity (NTLI) in humans were investigated using a newly developed, specific radioimmunoassay. Plasma NTLI was determined after its extraction with acid/acetone (recovery 77 +/- 4%). The intraassay and interassay coefficients of variation were 3.6% and 8.9%, respectively. The plasma concentration of human NTLI in normal subjects was 5.6 +/- 2.9 pmol/l and showed no significant sex difference. Ingestion of a test meal (150 g of Campbell's condensed meat soup) caused a biphasic rise in plasma NTLI from a basal level of 5.7 +/- 1.0 pmol/l to 10.8 +/- 1.2 pmol/l after 30 min and 9.6 +/- 1.1 pmol/l after 120 min. Ingestion of 5.5 g fat resulted in a biphasic rise in plasma NTLI from a basal level of 4.8 +/- 0.3 pmol/l to 8.9 +/- 0.3 pmol/l after 15 min and 11.9 +/- 0.4 pmol/l after 90 min. When 100-150 mg of ileal mucosa was perfused with a solution of 2 mEq/l fatty acids, 1 mM or 5 mM sodium taurocholate or 154 mM sodium bicarbonate (NaHCO3), the release of NTLI from the mucosa into the perfusate was 2.3-fold, 4.3-fold and 7.5-fold, respectively, over the base level. These results indicate that NTLI release is stimulated by ingestions of meat soup and fat and that NTLI present in the human ileum is released by the direct actions of solutions of fatty acid, sodium taurocholate, and NaHCO3 on the ileal mucosa. These findings strongly suggest that neurotensin (NT) has a physiological role in gut physiology.

Increase in neurotensin-like immunoreactivity in rat plasma after administration of calcium, bombesin and fat and its inhibition by somatostatin.

Neurotensin-like immunoreactivity (NTLI) is released from the small intestine into the blood after enteral administration of fat and systemic administration of bombesin. The purpose of the present investigation in the rat was to study the effect of intravenously (i.v.) administered CaCl2 on the plasma concentration of NTLI (p-NTLI) and to investigate the effect of i.v. infusion of somatostatin on basal NTLI release, as well as that stimulated by fat, bombesin and CaCl2. Administration of CaCl2 (5-25 mg X kg-1) increased p-NTLI levels in a significant and dose-dependent manner. In contrast, somatostatin 25-200 ng X min-1) reduced the basal p-NTLI levels, as well as the increase in p-NTLI levels induced by CaCl2 (10 and 25 mg X kg-1), fat (oleic acid, 0.5 ml) and bombesin (25 and 125 pmol X kg-1), in a dose-related manner. Injection of bombesin released immunoreactive material that was indistinguishable from synthetic bovine neurotensin (1-13) upon gel-filtration. This material seemed to be rapidly converted to the more stable metabolite neurotensin (1-8).

The use of a rat isolated ileal preparation to investigate the release of neurotensin.

An isolated and perfused preparation of rat ileum was used to investigate the effects of cholinergic, adrenergic and bombesin stimuli on neurotensin release into the vascular compartment. A vigorous release of neurotensin like immunoreactivity (NTLI) to 200% above basal values in response to intraluminal infusions of emulsified soybean oil (Intralipid) demonstrated the physiological responsiveness of the preparation. Carbachol significantly stimulated the release of both NTLI and bombesin like immunoreactivity (BLI), with maximal responses at 5 X 10(-9) mol/l carbachol of 100% and 400% above basal values for NTLI and BLI, respectively. Noradrenaline at 10(-6) and 10(-4) mol/l caused no significant release of NTLI but markedly inhibited spontaneous BLI release. Synthetic amphibian bombesin caused a marked release of NTLI to 81% and 100% above basal at infusion concentrations of 5 X 10(-11) and 5 X 10(-10) mol/l respectively. These results suggest that there is either a direct effect of cholinergic agents on the N cells bringing about NTLI release, or an indirect effect via the release of bombesin like peptides from intrinsic gut neurones. This preparation provides a useful model to study the complex neural, paracrine and endocrine interactions in the gastrointestinal tract.

Occurrence, storage and release of neurotensin-like immunoreactivity from the adrenal gland.

In the present study we have investigated the presence of radioimmunoassayable neurotensin-like immunoreactivity (NTLI) in extracts of adrenal glands from hen, guinea-pig, rat, cat, dog and man. In addition, gel filtration chromatography and studies on the storage site of NTLI, using subcellular fractionation techniques, were performed. Finally, NTLI release in relation to noradrenaline (NA) from cat adrenal glands upon splanchnic nerve activation was studied in vivo. In all species investigated except guinea-pig and man, the NTLI levels in the adrenal glands were higher than the levels in the heart (control tissue) but lower than in the ileum. Gel filtration of NTLI from cat adrenal glands revealed presence of roughly equal amounts of the whole neurotensin molecule, i.e. NT(1-13), and its main metabolite the N-terminal octapeptide NT(1-8), while in plasma from the adrenal vein almost exclusively NT(1-13) was revealed after electrical stimulation of the splanchnic nerve. The release of NTLI and NA upon splanchnic stimulation was antagonized by hexamethonium. Subcellular fractionation of cat adrenal glands indicated that NTLI was stored in a large subcellular organelle, co-migrating with chromaffin granules. We suggest that NT may be a new adrenal gland hormone or transmitter, possibly co-stored with NA in chromaffin cells. However, at the present stage it cannot be distinguished whether NT exerts its actions locally within the adrenal gland or after release into the systemic circulation.

The importance of bile and pancreatic juice for fat-induced release of neurotensin-like immunoreactivity (NTLI) from the small intestine of the rat.

The aim of the present study in the rat was to localize the gastrointestinal site(s) of NTLI release and to investigate the importance of bile and pancreatic juice for fat-induced NTLI release. Administration of Intralipid (2 ml) into the stomach and oleic acid (0.5 ml) into the duodenum increased the plasma concentration of NTLI (p-NTLI). The increase in p-NTLI levels occurred only when the jejunum and ileum were exposed to Intralipid but not when the exposure was limited to the stomach and duodenum. Exclusion of pancreatic juice significantly reduced the p-NTLI response and exclusion of both bile and pancreatic juice completely abolished the p-NTLI response to duodenally-administered oleic acid. The results indicate that neurotension is released by a direct luminal exposure of the neurotensin containing N-cells to fatty acids. It seems probable that fatty acids have to be transformed to a micellar form in order to release NTLI.

Cholinergic inhibition of meal stimulated plasma neurotensin like immunoreactivity in man

Meal stimulated plasma neurotensin like immunoreactivity (NTLI) was compared during saline or atropine infusion in six volunteers over six hours. Plasma gastrin and pancreatic polypeptide were also measured to compare the timing of their release to that of NTLI. Like plasma gastrin and PP, plasma NTLI rose rapidly following the meal, rising from 27±7 pmol/1 to a peak of 45±8 pmol/1 at 20 minutes (p < 0.05). Also, like that of pancreatic polypeptide, the release of NTLI was biphasic. Sixty minutes after the meal, plasma NTLI had returned to basal values, followed by a rise to a prolonged peak of 64±10 pmol/1 between 90–180 minutes (p < 0.05) returning once more to basal values by 240 minutes. Following atropine, basal plasma NTLI fell from 22±4 pmol/1 to 11±2 pmol/1 (p < 0.05), but rose to basal levels 30–60 minutes after the meal, where it remained unaltered for the remainder of the study. We conclude that both basal and meal stimulated plasma NTLI are inhibited by cholinergic blockade. Further, the similar temporal relationship between plasma NTLI and pancreatic polypeptide in the late phase of the meal response, suggests that a component of NTLI may mediate part of the intestinal phase of pancreatic polypeptide release.

Increased concentration of neurotensin-like immunoreactivity (NTLI) in rat plasma after administration of bombesin and bombesin-related peptides (porcine and chicken gastrin-releasing peptides).

The present experiments in the rat were designed to study the effect of bombesin on plasma concentrations of neurotensin‐like immunoreactivity (NTLI), to localize the gastrointestinal site(s) of bornbesin‐induced NTLI release and to compare the effects of bombesin and mammalian and avian bombesin‐related peptides (porcine and chicken gastrin‐releasing peptides) on the plasma concentrations of NTLI. Tail vein injections of bombesin (7.8–500 pmol X kg‐l) increased plasma NTLI in a dose‐related fashion and a peak concentration was obtained one min after the injection. Bombesin was injected into the arteries supplying certain vascular beds. A significant increase in plasma NTLI was found only after injection into the cranial mesenteric artery—the artery which supplies the intestinal area containing the highest concentration of NTLI. lntralurninal instillation of bombesin (1.25 nmol × kg‐1) into the distal part of the small intestine had no effect upon plasma NTLI. but intraperitoneal injection of bombesin (1.25 nmol × kg‐1) caused a significant increase in plasma NTLI. Intravenous injection of natural porcine and chicken gastrin‐releasing peptides and of synthetic porcine gastrin‐releasing peptide caused similar increases in plasma NTLI as bombesin. Chronic bilateral vagotomy did not change the effect of bombesin on plasma NTLI. The results show that the administration of bombesin and mammalian and avian bombesin‐related peptides markedly elevates the plasma concentration of NTLl in the rat and suggest that this elevation is largely due to release of NTLI from the area of the intestine supplied by the cranial mesenteric artery, i.e. jejunum and ileum.

Enhanced Neurotensin-Like Immunoreactivity Release in Total Gastrectomized Patients after 50 g OGTT

Neurotensin, originally isolated from the bovine hypothalamus (Carraway and Leeman 1973) is found not only in several parts of the brain (Carraway and Leeman 1976a) but also in the lower intestine (Orci, Baetens, Rufener, Brown, Vale and Guillemin 1976). Although neurotensin has a wide range of pharmacological effects (Carraway, Demer and Leeman 1976b), the concentrated localization of this peptide in the gut suggests that neurotensin may play a pathophysiological role in the gut disease. Recently, Bloom, Blackburn, Ebeid, Ralphs and Polak (1978) proposed the hypothesis that neurotensin may play an important role in the pathogenesis of dumping syndrome, based on measurement of plasma neutrotensin levels in gastrectomized patients. To examine the hypothesis, we compared changes of plasma neurotensin levels after 50 g OGTT in total gastrectomized patients with those in control subjects. In this paper, we report on enhanced neurotensin like immunoreactivity (NTLI) release in gastrectomized patients after 50 g OGTT.

Release of neurotensin-like immunoreactivity by intraduodenal oleic acid in man and inhibition of pentagastrin stimulated gastric acid secretion

Release of neurotensin-like immunoreactivity by intraduodenal oleic acid in man and inhibition of pentagastrin stimulated gastric acid secretion

The effect of ingestion of amino acids, glucose and fat on circulating neurotensin-like immunoreactivity (NTLI) in man.

The effect of ingestion of amino acids (Vamin N), glucose, and fat (Intralipid) on the concentration of neurotensin-like immunoreactivity (NTLI) in plasma was determined in 6 healthy male volunteers. After ingestion of Intralipid (55 ml, 200 mgxml-1) there was a significant increase in the plasma concentration of NTLI. The calculated integrated total NTLI response (6.7 +/- 2.5 nM over 180 min) was statistically significant. Although isocaloric amounts of Vamin and glucose also increased the plasma concentration of NTLI, these increases were not statistically significant. The data indicate that fat is an important stimulus for the release of NTLI from the small intestine. It is suggested that neurotensin, or a metabolite, in blood may be a hormone involved in the postprandial inhibition of gastric motility and gastric acid secretion elicited from the small intestine.