Edwin J. Draviam

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.

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)

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 measuredin 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-Tyro-BBS in a dose-dependent manner, while structurally unrelated peptides did not compete for the 125I-Tyro-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) > GRP (1–27) > neuromedin B > BBS. The BBS-receptor antagonists, [Leu13-ψ-(CH2NH) Lcu14]-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. The inhibitory potency of D-Phe6,BN[6-13]PA was significantly greater than that of LL-1313S. Molecular mass of the specific binding proteins for BBS/GRP was determined to be 70–80 KDa, by chemical cross-linking methods. The 70-80 KDa binding proteins were specific for binding GRP-related peptides and were displaced in dose-dependent manner by increasing doses of BBS. These results thus suggest that the colonic mucosa may be yet another target for GRP related peptides.

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 (NT1–3) 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 aminoterminal and carboxyterminal 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.

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<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<0.05) elevation of plasma levels of secretin.

A high pressure liquid chromatography-radioimmunoassay method for measurement of cholecystokinin-8 and cholecystokinin-33/39 in plasma

Abstract A new high pressure liquid chromatography-radioimmunoassay method is described for the measurement of cholecystokinin-8 (CCK-8) and CCK-33/39 in plasma. The plasma levels of CCK-8-sulfate, CCK-8-desulfate, and CCK-33/39 were measured during the infusion of intraduodenal fat.