A human gastric cancer cell line possesses a functional receptor for gastrin-releasing peptide.

Bombesin (BBS) exhibits diverse biological functions including those of neurotransmitter, regulator of gastrointestinal hormone release, and mitogen. Gastrin-releasing peptide (GRP, the mammalian equivalent of BBS) is found in mucosal cells of the gastric fundus and antrum. We determined whether a human gastric cancer cell line (SIIA) expresses a functional GRP-receptor (GRP-R). BBS increased intracellular calcium ([Ca2+]i), and a specific GRP-R antagonist, ([D-Phe6, Des-Met14]-BBS (6-14)-ethylamide), blocked BBS-induced increase in [Ca2+]i. SIIA cells possess GRP-R mRNA by reverse transcriptase-PCR. Furthermore, these cells possess an 80-kDa cell surface protein that specifically binds BBS with two high-binding affinities (Kd1 = 0.6 nM, Kd2 = 6.7 nM). These findings indicate that SIIA cells possess a GRP-R that is capable of physiological signal transduction, though the cellular response remains unknown.

Neurotensin expression and release in human colon cancers.

Neurotensin (NT), a distal gut peptide released by intraluminal fats, is trophic for normal small bowel and colonic mucosa. In addition, NT stimulates growth of certain colon cancers; the mechanism for this effect is not known. The purpose of this study was to determine whether human colon cancers (HCC) (1) express the mRNA for NT/neuromedin N (N), (2) produce NT peptide, and (3) express the mRNA for a functional NT receptor (NTR). RNA was extracted from four HCC cell lines in culture, nine HCC lines established in athymic nude mice, and from six HCC and adjacent normal mucosa from freshly resected operative specimens; the RNA was analyzed for NT/N mRNA by Northern hybridization with a complementary DNA probe. Neurotensin peptide content, NTR expression, and intracellular Ca++ ([Ca++]i) mobilization in response to NT were evaluated in three HCC cell lines (LoVo, HT29, HCT116). Neurotensin/N mRNA transcripts were identified in all four of the HCC cell lines and in one of nine HCC in nude mice. Neurotensin expression was found in two of six freshly resected HCC and in none of the six corresponding samples of normal mucosa. Neurotensin peptide was identified by RIA in LoVo, HT29, and HCT116. In addition, NTR mRNA was found in HT29 and HCT116. Neurotensin stimulated [Ca++]i mobilization in HCT116 (without serum) and in LoVo (with 0.25% serum). These findings demonstrate the presence of NT/N mRNA and NT peptide and the presence of a functional NTR in certain HCC. Neurotensin, a potent trophic factor for normal gut mucosa, may function as an autocrine growth factor in certain human colon cancers.

Intravenous amino acids stimulate human gallbladder emptying and hormone release.

Gallbladder stasis during prolonged total parenteral nutrition (TPN) has been documented. We have examined the effect of intravenous amino acid infusion on human gallbladder contraction and release of cholecystokinin (CCK). Five healthy adult volunteers were given amino acid infusions at different rates (65, 125, 240, and 600 mg.kg-1.h-1). The volume of the gallbladder was calculated by means of ultrasonographic measurements. Plasma samples were analyzed for CCK immunoreactivity. Gallbladder and hormone responses after intravenous amino acids were compared with responses after a fat meal, after a protein meal, and after ingestion of an oral amino acid mixture. We found that intravenous amino acids stimulated human gallbladder contraction in a dose-related manner. The mechanism of stimulation may be through the release of CCK although significant correlation was not demonstrated. The magnitude of response is similar to that seen after meal stimulation. To compare the delivery of amino acids during a standard meal and during each dose of intravenous amino acids, peripheral plasma levels of dietary amino acids were measured after a standard commercially prepared enteral supplement meal and after each dose of intravenous amino acids. Our lower doses of amino acid infused resulted in levels of circulating amino acid comparable to those after a meal. The induction of gallbladder contraction and release of CCK in human recipients of parenteral nutrition may be of value in some circumstances.

Effect of total jejunoileal denervation on fat-stimulated release of peptide YY and cholecystokinin.

We have investigated the effect of total denervation of the jejunoileum (JDNv) on stimulated release of peptide YY (PYY) and cholecystokinin-33/39 in five dogs prepared with chronic gastric and duodenal cannulas. JDNv was performed by stripping the adventitia from the superior mesenteric artery and vein, transecting the small bowel mesentery, and division (with reanastomosis) of the small bowel at the ligament of Treitz and ileocecal junctio. Introduodenal corn oil (3 ml/kg/hr) was given before JDNv and 1 and 2 months after JDNv. Intravenous bombesin (400 pmol/kg/hr) was given (on nonconsecutive days) before JDNv and 1 month after JDNv. Plasma PYY and cholecystokinin levels were measured by specific radioimmunoassay. Release of PYY was enhanced after JDNv. The integrated release of PYY (ng.[0 to 60 min]/ml) after intraduodenal corn oil was as follows: before JDNv, 4.1 +/- 1.2; 1 month after JDNv, 16.0 +/- 2.7; and 2 months after JDNv, 10.3 +/- 2.2. Similar results were noted with intravenous bombesin (3.7 +/- 0.9 [before JDNv] vs 12.0 +/- 0.7 [1 month after JDNv]). Corn oil-stimulated release of cholecystokinin was abolished after JDNv (before JDNv 2.2 +/- 1.1; 1 month after JDNv, 0.6 +/- 0.3; and 2 months after JDNv, 0.4 +/- 0.6). Basal plasma levels of PYY and cholecystokinin were not affected by JDNv. We conclude that JDNv enhances PYY and abolished cholecystokinin release, which provides evidence for different mechanisms of neural control.

Protective action of luminal bile salts in necrotizing acute pancreatitis in mice.

Bile salts in the intestinal lumen act to inhibit the release of cholecystokinin (CCK). Recent studies have shown that CCK may play a permissive role in the development of acute pancreatitis. In this study, the amount of luminal bile salts in female Swiss Webster mice was either decreased by feeding 4% (wt/wt) cholestyramine or increased by feeding 0.5% sodium taurocholate for 1 wk. Plasma levels of CCK were stimulated by cholestyramine and inhibited by taurocholate. Then, acute pancreatitis was induced either by caerulein injections, or by feeding a choline-deficient, ethionine-supplemented (CDE) diet. Feeding of cholestyramine significantly decreased survival from 25% to 0% in the CDE pancreatitis, and increased the magnitude of elevation of serum amylase levels and the extent of pancreatic necrosis in both models of pancreatitis; CCK-receptor blockade with CR-1409 completely abolished the adverse effects of cholestyramine. In contrast, feeding of taurocholate significantly increased survival to 100% and decreased the elevation of serum amylase and pancreatic necrosis; CCK-8 antagonized these actions of taurocholate. Luminal bile salts appear to provide a physiologic protection against necrotizing pancreatitis, at least in part, both by inhibiting the release of CCK and by promoting resistance of the pancreas to CCK excessive stimulation in vivo.

Effect of oral fat on plasma levels of neurotensin and neurotensin fragments in humans. Characterization by high-pressure liquid chromatography.

The effect of ingestion of fat (Lipomul 1 g/kg) on the circulating levels of neurotensin (NT1-13) and amino-terminal fragments (NT1-8, NT1-11) and carboxy-terminal fragment (NT8-13) of NT were investigated in six healthy male volunteers. NT and NT fragments were extracted from plasma collected at 0, 15, 30, and 60 min after ingestion of fat, and the plasma levels of NT1-13 and NT fragments were characterized using high-pressure liquid chromatography and radioimmunoassay techniques. Significant elevations of plasma levels of NT1-8, NT1-11, and NT1-13 were observed at 15, 30, and 60 min after fat ingestion. The maximum elevations were 273% for NT1-8, 234% for NT1-11, and 54% for NT1-13. NT8-13 levels failed to rise significantly when compared to basal levels. These findings indicate that both the amino-terminal and carboxy-terminal fragments of NT are either released along with intact NT or are formed as metabolites from NT1-13 in response to ingestion of fat in man.

Involvement of cholecystokinin receptors in the adverse effect of glucocorticoids on diet-induced necrotizing pancreatitis.

The mechanism that explains the association between corticoids and acute pancreatitis is unknown. Our hypothesis was that chronic glucocorticoid treatment could adversely affect the course of hemorrhagic pancreatitis by acting through cholecystokinin (CCK) receptors. Acute necrotizing pancreatitis was induced by feeding young female mice a choline-deficient, ethionine-supplemented (CDE) diet for 60 hours. Treatment with hydrocortisone (10 mg/kg/day) was begun 1 week before pancreatitis. At the onset of the CDE diet, a group of hydrocortisone-treated mice were also given the CCK receptor antagonist CR-1409 (5 mg/kg three times a day). Control mice received injections of saline solution. A follow-up of 336 hours was conducted for survival analysis. Hydrocortisone given alone did not produce pancreatitis. Hydrocortisone, however, did increase the pancreatic necrosis caused by the CDE diet (from 40% to 70%) and significantly reduce survival (from 40% to 9%). CR-1409 completely abolished the adverse effects of hydrocortisone on pancreatitis. We measured amylase release by dispersed pancreatic acini from mice chronically treated with hydrocortisone in response to CCK-8. Treatment with hydrocortisone increased both the sensitivity and the responsiveness of the pancreas to CCK-8. We conclude that glucocorticoids alone may not induce acute pancreatitis, but they can increase the risk of a more severe form of pancreatitis developing. The glucocorticoid effect appears to be attributable to a CCK receptor-mediated sensitization of the pancreas to endogenous CCK. Thus, CCK-receptor blockade may improve survival in necrotizing pancreatitis associated with chronic glucocorticoid treatments.

Bile inhibits release of cholecystokinin and neurotensin.

We have investigated the effect of bile on fat-stimulated release and basal plasma levels of cholecystokinin-33/39 (CCK) and neurotensin in six awake dogs prepared with chronic gastric and duodenal cannulas. Experimental bile diversion was achieved by catheterization of the common bile duct through the duodenal cannula; the gallbladder was undisturbed. Bile diversion significantly enhanced the release of both CCK and neurotensin that was stimulated by intraduodenal (ID) infusion of a triglyceride suspension (corn oil) (0.5 gm/kg-hr). The integrated release with ID triglyceride (ng [0 to 90 min]/ml) for CCK was control 5.58 +/- 0.83, bile diversion 14.47 +/- 2.81, bile excess 1.68 +/- 0.56, and for neurotensin was control 0.35 +/- 0.19, bile diversion 1.26 +/- 0.35, and bile excess 0.45 +/- 0.31. ID infusion of excessive bile (bile collected during bile diversion) significantly inhibited both the release and basal levels of CCK. Bile diversion alone did not modify plasma levels of CCK or neurotensin. We conclude that: endogenous bile exerts a negative feedback effect on release of CCK and neurotensin induced by triglyceride and on basal plasma levels of CCK; bile is unnecessary for the stimulation of endocrine cells in the intestinal mucosa by dietary fat; and measured basal levels of CCK and neurotensin represent a real amount of circulating peptide in the fasting state, that is, the basal levels are real and not artifactual.

Effect of aging on gallbladder contraction and release of cholecystokinin-33 in humans.

The objective of this study was to examine the effect of aging on gallbladder contraction and cholecystokinin (CCK) release, as well as on the correlation between the two in humans who are free of gallbladder disease. Twenty-nine human volunteers were divided into a young group of 14 individuals (ages 22 to 42 years, median age 32 years) and an older group of 15 individuals (ages 60 to 84 years, median age 66 years). In the study each person in both groups was given corn oil (Lipomul), 1.5 ml/kg, by mouth after an overnight fast. Blood was collected for measurement of CCK-33 by radioimmunoassay before and at intervals after ingestion of Lipomul. Simultaneous measurements of gallbladder volume were obtained by real-time varian ultrasonography. Both fasting and fat-stimulated concentrations of CCK in plasma were significantly higher in the older individuals than in the younger volunteers. The 60-minute integrated measurement of CCK release was significantly increased in the older people as compared with the young. Both fasting and maximally contracted gallbladder volumes were equal in the older and younger groups. The rate of emptying of the gallbladder was equal in both age groups, but the gallbladders of older people appeared to show an earlier initiation of contraction. The highly significant correlation of gallbladder contraction with levels of CCK was similar in both age groups, but the sensitivity of the gallbladder to CCK in the older people was significantly decreased. In conclusion, both fasting and fat-stimulated plasma levels of CCK increase with aging. The sensitivity of the gallbladder muscle to stimulation by CCK is diminished with age, but this appears, teleologically, to be matched by the increased release of CCK, so the kinetics of gallbladder emptying are little different in the aged.

Importance of the ileum in neurotensin released by fat.

Neurotensin is a potent stimulant of pancreatic exocrine secretion. Ileal mucosa is the storage site for about 90% of total neurotensin. Release occurs rapidly after a fatty meal and during perfusion of the duodenum and jejunum with fat but not during perfusion of the ileum with fat. To determine the origin of neurotensin released after fat stimulation, we studied the pattern of release of neurotensin before and after resection of the distal two thirds of the small bowel. Six dogs with gastric and duodenal fistulas were studied on different days. All dogs received infusions (in random order) of intraduodenal corn oil (Lipomul) (3 ml/kg/hr) and intravenous calcium chloride (0.36 mmol/kg intravenous bolus, followed by 0.36 mmol/kg/hr infusion) before and 6 weeks after resection of the distal two thirds of the small bowel with preservation of the ileocecal valve. Plasma levels of neurotensin were measured by specific radioimmunoassay. We found that release of neurotensin, in response to both intraduodenal Lipomul and intravenous calcium chloride stimulation, was abolished by resection of the distal small bowel. Before surgery, Lipomul-stimulated release of neurotensin rose to a peak concentration of 51 +/- 17 pg/ml at 30 minutes. After surgery there was no release (the levels were unchanged from basal). Before surgery, intravenous calcium chloride produced a peak release of neurotensin (52 +/- 15 pg/ml) 2 minutes after bolus injection. After surgery, neurotensin was not released by intravenous calcium. We conclude that the source of neurotensin released by perfusion of the proximal gut and by intravenous calcium infusion is the ileum. The release of neurotensin from the distal gut appears to be dependent on a signal from proximal to distal gut. The identity of the signal is unknown but is either a nerve reflex or a peptide agent.

The role of neurotensin in human gallbladder motility.

Gallbladder contraction in response to a fatty meal is thought to be caused by release of cholecystokinin (CCK). We have previously demonstrated a close correlation between circulating concentrations of CCK and contraction of the gallbladder in normal humans and in gallstone patients. Recent studies in animals, however, have shown that other potentially cholecystokinetic hormonal agents are released by a fatty meal, which suggests that other hormones may be involved in postprandial gallbladder contraction. Neurotensin, a 13-amino acid peptide, is released by fat; we have shown it to cause gallbladder contraction in dogs. In the present study, we measured release of neurotensin in seven normal adult volunteers. We determined the effects of infused neurotensin (4 pmol/kg-min) on gallbladder contractility, measured by ultrasonography in 10 adult volunteers, and we evaluated release of neurotensin in eight patients with gallstones. After ingestion of fat, we found significant release of neurotensin in normal volunteers from a mean basal concentration of 15.9 +/- 3.5 pg/ml to a maximum of 34.7 +/- 0.2 pg/ml. In the gallstone patients after fat ingestion, neurotensin rose from a basal of 16.8 +/- 3.1 pg/ml to a maximum of 53.4 +/- 28.1 pg/ml, which was a significantly greater release than in controls. Intravenous infusion of neurotensin produced dilatation of the gallbladder (from a mean basal volume of 13.7 +/- 2.3 cc to 20.0 +/- 1.8 cc). Neurotensin causes relaxation of the gallbladder in humans and, by contributing to stasis, may be involved in the formation of gallstones.

Role of neurotensin in pancreatic secretion.

The objective of this study was to examine the effect of neurotensin (NT) on pancreatic exocrine secretion in awake dogs (n = 5) with chronic gastric and pancreatic fistulas. Intravenous (IV) infusion of NT (1 microgram/kg/hr) alone significantly stimulated pancreatic secretion of protein and bicarbonate without causing release of secretin or cholecystokinin-33 (CCK-33). IV NT potentiated the secretory response of pancreatic bicarbonate to the intraduodenal (ID) infusion of HCl alone and to ID infusions of the amino acids, phenylalanine and tryptophan (AA) alone, as well as to an ID mixture of AA plus HCl. IV NT acted in an additive manner with ID AA, ID HCl, or ID AA plus HCl in the stimulation of pancreatic protein output. The addition of IV NT to each luminal secretagogue (ID AA, ID HCl, or ID AA plus HCl) failed to elevate plasma concentrations of CCK-33 or secretin over those observed during ID infusion of each secretagogue alone. ID corn oil (Lipomul) stimulated the simultaneous release of CCK-33, NT, and secretin significantly; IV infusion of NT (0.5 microgram/kg/hr) resulted in plasma NT levels that were similar to levels observed after ID Lipomul. These studies provide evidence that endogenous NT, CCK, and secretin may interact in the physiologic regulation of pancreatic exocrine secretion.

High dose methotrexate therapy of solid tumors: observations relating to clinical toxicity.

In clinical studies performed during 111 infusions of high dose methotrexate (MTX) we have evolved a clinical and laboratory protocol which permits such therapy without prohibitive risk to the patient. The plasma MTX data obtained indicate that pharmacokinetic disposition is dose related during these infusions and that such data are useful in identifying patients at risk from serious toxicity.