Early stages of gallstone formation in guinea pig are associated with decreased biliary sensitivity to cholecystokinin.

The purpose of this study was to measure differences in gallbladder sensitivity to cholecystokinin (CCK) in vivo during the early stages of gallstone formation and to correlate these findings to gallbladder CCK receptors. Guinea pigs were placed on either a normal diet or a two-week cholelithogenic diet, after which gallbladder emptying pressure to exogenously administered CCK was measured in vivo, according to the presence or absence of gallstones. At all doses of CCK tested (except 10(-10) mol/kg), the gallbladder response to CCK of guinea pigs that did not develop gallstones (on the cholelithogenic diet) was more sensitive than that of guinea pigs that did develop gallstones. Neither group was different from guinea pigs on a normal diet. In a second experiment, CCK receptors were measured on gallbladder muscularis from guinea pigs after two weeks on the same diet as in the first experiment. Those guinea pigs that did not develop gallstones had greater concentrations of CCK receptors (149 +/- 9 fmol/mg protein) than those that did develop gallstones (70 +/- 23 fmol/mg protein). Neither group was different from normal diet guinea pigs (119 +/- 57 fmol/mg protein). At the time point measured, there were no differences in the lipid chemistry or protein concentrations of gallbladder bile between the guinea pigs on the cholelithogenic diet that did or did not develop gallstones, or those on normal guinea pig chow. We conclude that the early stages of gallstone formation in guinea pigs are associated with decreased gallbladder sensitivity to CCK and that this change may be due to a lower concentration of CCK receptors on the gallbladder smooth muscle.

High-affinity binding sites for bombesin on mouse colonic mucosal membranes.

Bombesin (BBS) has specific biological effects on colonic mucosal cells, but the presence of BBS receptors on colonic mucosa have not been described to-date. In the present study we examined the mouse colonic mucosal membranes for the presence of specific binding sites for BBS/gastrin releasing peptides (GRP), and characterized the binding kinetics and molecular weight of the specific binding proteins. The radiolabeled ligand (125I-Tyr4-BBS), in the absence or presence of a 1000-fold excess of BBS, was used to establish the optimal binding assay conditions of time, pH and temperature for measuring the maximum number of specific binding sites for BBS related peptides. Under the optimal binding assay conditions, BBS displaced the binding of 125I-Tyr4-BBS in a dose-related manner. A single class of high-affinity binding sites (Kd = 0.23 +/- 0.02 nM) for BBS were measured, with a binding capacity of 27.3 +/- 4.6 fmoles/mg membrane protein. The binding sites were specific for binding BBS/GRP related peptides, since all structurally related peptides inhibited the binding of 125I-Tyr4-BBS in a dose-dependent manner, while structurally unrelated peptides did not compete for the 125I-Tyr4-BBS binding sites. The relative binding affinity (RBA) of BBS/GRP related peptides was determined to be in the order of GRP (14-27) = GRP (18-27) greater than GRP (1-27) greater than neuromedin B greater than BBS. The BBS-receptor antagonists, [Leu13-psi-(CH2NH) Leu14]-BBS (LL-BBS) and D-Phe6, BN(6-13) propylamide (D-Phe6, BN(6-13)-PA), inhibited the specific binding of 125I-Tyr4-BBS to colonic mucosal membranes in a dose-dependent manner.(ABSTRACT TRUNCATED AT 250 WORDS)

Expression of cholecystokinin forms by medullary thyroid cancer.

We have studied the production and release of cholecystokinin (CCK) forms by rat medullary thyroid cancer (MTC) in vivo. MTC cells were inoculated s.c. into 8 Wag-Rij rats. One month later, after i.v. injection of calcium plasma levels of CCK, calcitonin and tissue contents of gastrin and calcitonin were determined by radioimmunoassay (RIA), immunocytochemistry, and high-pressure liquid chromatography (HPLC). The tumors were passed 4 times to 8 rats in 1-month intervals fasting levels of CCK in control rats were unaffected by calcium stimulation, and in tumor-bearing rats, plasma CCK was elevated in 3 out of 4 passages, falling to normal levels at the end of passage 4. Hypercalcemia had no effect on plasma levels of CCK in tumor-bearing rats, but did stimulate the release of calcitonin in both control and some tumor-bearing rats in later passages. CCK-8 sulfate was found in all 4 tumor passages but not CCK-33/39. We conclude that rat MTC synthesizes and releases CCK-8, but unlike calcitonin, release of CCK appears unresponsive to calcium stimulation.

Characterization of secretin release in response to food and intraduodenal administration of fat and hydrochloric acid.

The development and validation of a radioimmunoassay that detects release of secretin in plasma in response to low doses of secretagogues [intraduodenal HCl (0.033 meq/min); intraduodenal sodium oleate (0.04 mmol/min)] or an oral mixed meal in conscious dogs is described. Plasma secretin levels increased significantly (P less than 0.05) in response to an oral mixed meal in conscious dogs from a basal level of 4.0 to a peak level of 12.3 pg/ml at 15 min. Infusion of graded doses of HCl (2, 4, 8, 16, meq/hr for 30 min) intraduodenally in six dogs resulted in significant elevation of plasma secretin levels in a dose-dependent manner. The pancreatic bicarbonate and volume outputs correlated with the dosage of HCl administered and with the elevations in plasma secretin concentrations. Intraduodenal infusion of increasing doses of sodium oleate (2.4, 4.8, 9.6, and 19.2 mmol in 15-min periods) resulted in a significant (P less than 0.05) elevation of plasma levels of secretin.

Molecular characterization of bombesin receptors on rat pancreatic acinar AR42J cells.

A biologically active, chemically defined, radioactive ligand was used for characterizing bombesin (BBS) receptors on rat pancreatic acinar cancer cells (AR42J). [Tyr4]BBS, iodinated with enzymobeads and fractionated by high-performance liquid chromatography, was monitored for biological activity as evidenced by gastrin release from perfused isolated rat stomach. The monoiodinated peptide peak was greater than 95% biologically active, with a specific activity of greater than 2,000 disintegrations.min-1.fmol-1. The maximum number of BBS receptors per cell were measured at 30 degrees C after 20-25 min of incubation; binding was submaximum at temperatures lower or higher than 30 degrees C. A single class of high-affinity binding sites (Kd = 1.77 +/- 0.21 nM) was identified for BBS on AR42J cells and nonspecific binding was less than 20-30% at all points. A total of 1.47 +/- 0.14 x 10(5) specific BBS binding sites per cell were measured that were specific for BBS and gastrin-releasing peptide (GRP) analogues. Iodinated GRP-(1-27) was cross-linked to BBS receptors on AR42J cells using several bifunctional cross-linking reagents followed by polyacrylamide gel electrophoresis of the solubilized receptor complex under reducing and nonreducing conditions. A densitometric analysis of the autoradiographs demonstrated the presence of an approximately 80- to 85-kDa molecular form of the receptor as a major component under both reducing and nonreducing conditions. These results indicated that the receptor molecule is a single subunit without multiple chains covalently attached by disulfide bonding.

Effect of age and sensitivity to cholecystokinin on gallstone formation in the guinea pig.

The purpose of this study was to evaluate the effect of age and the role of cholecystokinin therapy on gallstone formation in guinea pigs. Guinea pigs (31 1-mo-old, 31 1-yr-old, and 23 3-yr-old) were placed on a cholelithogenic diet for 2 wk while another 10 guinea pigs of each age group remained on regular chow. Half of each group received a daily injection of cholecystokinin (0.5 nmol/kg). After 2 wk, guinea pigs were killed and the gallbladders were examined for gallstones. The concentrations of bile constituents were determined. The prevalence of gallstones was: 1-mo-old, control 0 out of 16, cholecystokinin 1 out of 15; 1-yr-old, control 3 out of 14, cholecystokinin 5 out of 16; 3-yr-old, control 10 out of 11, cholecystokinin 3 out of 8. Gallstone formation was significantly greater in 3-yr-old controls than in the two younger control groups, and cholecystokinin treatment significantly reduced the incidence of gallstones to near the level seen in younger guinea pigs. In the two younger age groups (but not in the 3-yr-old group), the cholelithogenic diet significantly reduced the concentration of bile salts in bile below that of guinea pigs on a normal diet. The cholelithogenic diet and treatment with cholecystokinin did not alter the relative compositions of bile lipids from that of guinea pigs on a normal diet in any of the three ages studied. In the second experiment we measured gallbladder emptying in response to exogenous infusion of cholecystokinin-8 (100 fmol/kg/h-100 nmol/kg/h) in the same three age groups of guinea pigs in vivo that had been maintained on regular chow. There was no difference in cholecystokinin sensitivity between the two younger age groups, but both were significantly more sensitive to cholecystokinin than the 3-yr-old guinea pigs in rate of gallbladder emptying in the dose range 1 pmol/kg/h-1 nmol/kg/h. We conclude that a major factor in the increased incidence of gallstone formation in the aged guinea pig gallstone model is decreased gallbladder emptying due to decreased gallbladder sensitivity to cholecystokinin.

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.

Differential effects of Ca2+ on proliferation of stomach, colonic, and pancreatic cancer cell lines in vitro.

Calcium intake inhibits growth of colon cancer in vivo, the mechanisms of which are not fully elucidated. The objective of this study was to determine whether Ca2+ directly affects the growth of colon cancer cells in vitro and to compare the effects of Ca2+ on the growth of several gastroenteropancreatic cancer cells, including mouse colon cancer (MC-26), human colon cancer (LoVo and WIDR), human gastric cancer (AGS and SII), and human pancreatic cancer (PANC-1 and MIA) cells. All tumor cell lines tested grew in medium containing low concentration (approx 0.16 mM) of Ca2+. Higher concentrations of Ca2+ significantly inhibited the growth of all three colon cancer cell lines tested but had no significant effect on proliferation of the stomach and pancreatic cancer cell lines. Growth of AGS cells, in the presence of 0.1 or 0.5 mM EGTA (resulting in the loss of the extracellular Ca2+) was similar to that observed in the absence of EGTA, indicating that AGS cells were relatively insensitive to loss of extracellular Ca2+. In the presence of TMB-8, an inhibitor of intracellular Ca2+ release, the growth of colonic cancer cell lines was inhibited in a dose-dependent manner, indicating that a minimum basal level of intracellular Ca2+ was required for continued proliferation of colon cancer cells. The stomach cancer cell lines (AGS) was once again less sensitive to the effects of TMB-8 than were the colon cancer cells, indicating an inherent difference in Ca2+ requirements and sensitivity to Ca2+ for growth of different gastroenteropancreatic cancer cells in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)

Peptide YY inhibits the insulinotropic action of gastric inhibitory polypeptide.

Peptide YY (PYY) is released from the gut after ingestion of fat or after a meal. The purpose of this investigation was to examine the effect of PYY on gastric inhibitory polypeptide (GIP)-stimulated insulin release in conscious dogs with gastric and duodenal fistulas. In control experiments, 6 dogs received GIP (400 pmol/kg, i.v., for 1 h) and glucose (0.6 g/kg, i.v., for 1 h); the integrated insulin response over a 1-h period was 142 +/- 32.7 ng-60 min/ml. The plasma GIP levels achieved by this procedure were similar to those observed by intraduodenal infusion of Lipomul (2 ml/min), suggesting that the dose of GIP used was within the physiologic range. Intravenous infusion of three different doses of PYY (100, 200, or 400 pmol/kg.h) caused a significant inhibition of insulin release stimulated by GIP + glucose; the integrated insulin response was reduced to 105, 88, and 79 ng-60 min/ml, respectively. On the other hand, PYY (400 pmol/kg.h) had no effect on insulin secretion induced by intravenous glucose (0.6 g/kg.h) alone. These results indicate that PYY specifically inhibits the insulinotropic action of GIP and that PYY may play a negative-feedback regulatory role in the enteroinsular axis.

Development and age-related changes in pancreatic cholecystokinin receptors and duodenal cholecystokinin in guinea pigs.

We have investigated the changes associated with development and aging on the interrelationships between cholecystokinin (CCK) and the pancreas in the guinea pig. Three groups (1 month old, 1 year old, and 3 years old) of male guinea pigs were sacrificed while feeding in order to measure food-stimulated levels of CCK in blood and in duodenal mucosa by radioimmunoassay (RIA), as well as the pancreatic concentrations of CCK receptors. Systemic blood concentrations of CCK did not change with age. However, the concentration and content of CCK in duodenal mucosa increased more than 3-fold with age. A single class of high-affinity (KD less than or equal to 0.1 nM) CCK-receptor was found on the pancreatic membranes. The concentration (fmol/mg protein) of these receptors significantly diminished by one-half with increasing age. We also found an apparently similar fall in the receptor-binding affinity, but the difference was not significant. We conclude that in the guinea pig, duodenal content of CCK increases so as to compensate for the decreasing concentration of pancreatic CCK receptors, or, perhaps, vice versa. The diminished exocrine function of the pancreas, seen with increasing age, may well reflect both the diminished number of CCK-receptors and the reduction of pancreatic acinar cells.

Bile enhances release of insulin: an incretin-mediated effect.

Ingestion of food stimulates secretion of bile and release of gut hormones that enhance nutrient-stimulated release of insulin. The extent of physiologic participation of bile in the enteroinsular axis was examined in seven conscious dogs (weight: 20 +/- 2 kg) that were prepared for study with chronic cannulas placed in the duodenum opposite the ampulla of Vater. On separate days, a meal consisting of 10 gm (MG-10), 25 gm (MG-25), or 62 gm (MG-62) of glucose (dextrose) was given orally in the presence of normal bile flow or during bile diversion. Bile diversion was achieved by catheterization of the common bile duct via the duodenal cannula. The insulin responses (given as ng [0-120] min/ml) to the different glucose meals, with bile present (BP) or absent (BA) in the lumen, were as follows: MG-10, 81 +/- 8 BP and 72 +/- 11 BA; MG-25, 172 +/- 25 BP and 100 +/- 6 BA (p less than 0.05); and MG-62, 390 +/- 79 BP and 153 +/- 32 BA (p less than 0.05). Only MG-25 and MG-62 produced a significant elevation of plasma glucose concentrations. Release of gastrin was not affected by either the presence of bile or the glucose content of the meal. We conclude that endogenous bile enhances nutrient-stimulated release of insulin, this effect is glucose-dependent with a threshold of approximately 1 gm/kg of glucose, and bile may facilitate the release of insulinotropic hormones other than gastrin.

Quality control for a 14- to 18-megohm-cm deionized water supply.

We studied the quality control for a 14- to 18-megohm-cm deionized water system with multiple outlets. The quality of water was determined by measuring specific resistance, performing a total colony count, trace metal analysis, and high-performance liquid chromatographic analysis. The specific resistivity of water was at the level of 17 to 18 megohms-cm at the recirculation loop and greater than 10 megohms-cm at the individual faucets. The microbial content was consistently less than 100 colony-forming units/mL. Periodic trace-metal analysis indicated less than 1 microgram/L of copper, bismuth, zinc, cadmium, mercury, thallium, and lead. High-performance liquid chromatographic analysis, as determined by the absorbance of the highest peak by ultraviolet and fluorescence spectroscopy, indicated less than or equal to 0.001 absorbance unit. Our experience suggests that maintaining laboratory water at 14 to 18 megohms using a plastic system with plastic faucets eliminates the likelihood of bacterial organic and trace-metal contamination of the water.

Vapor pressure and freezing point osmolality measurements applied to a volatile screen.

This is a report of a rapid and precise screening procedure, developed for the determination of ethanol in serum using osmolality measurements. The osmolality of the patient is determined by freezing point method (freezing point osmometry) and dew point (water vapor pressure osmometry) method. The difference between freezing point osmolality and vapor pressure osmolality (delta osm) is due to the presence of volatiles in the serum, because the volatiles are not measured by vapor pressure osmometry. The amount of ethanol (mg/dL) in serum is estimated by multiplying delta osm by a factor of 4.2. As a comparison method, ethanol also is measured by a spectrophotometric alcohol dehydrogenase method. In addition, a significant difference between an osmometric alcohol assayed value and enzymatic spectrophotometric measurement indicates the presence of volatiles, other than ethanol. In addition to ethanol there is a linear relationship between osmolality and isopropanol or methanol when added in vitro to serum.