The gastrointestinal peptide cholecystokinin (CCK) causes the release of pancreatic digestive enzymes and growth of the normal pancreas. Exogenous CCK administration has been used in animal models to study pancreatitis and also as a promoter of carcinogen-induced or Kras-driven pancreatic cancer. Defining CCK receptors in normal human pancreas has been problematic because of its retroperitoneal location, high concentrations of pancreatic proteases, and endogenous RNase. Most studies indicate that the predominant receptor in human pancreas is the CCK-B type, and CCK-A is the predominant form in rodent pancreas. In pancreatic cancer cells and tumors, the role of CCK is better established because receptors are often overexpressed by these cancer cells and stimulation of such receptors promotes growth. Furthermore, in established cancer, endogenous production of CCK and/or gastrin occurs and their actions stimulate the synthesis of more receptors plus growth by an autocrine mechanism. Initially it was thought that the mechanism by which CCK served to potentiate carcinogenesis was by interplay with inflammation in the pancreatic microenvironment. But with the recent findings of CCK receptors on early PanIN (pancreatic intraepithelial neoplasia) lesions and on stellate cells, the question has been raised that perhaps CCK actions are not the result of cancer but an early driving promoter of cancer. This review will summarize what is known regarding CCK, its receptors, and pancreatic cancer, and also what is unknown and requires further investigation to determine which comes first, the chicken or the egg, "CCK or the cancer."

Nonsulfated CCK(58) [CCK(58)(ns)] has not been considered to be of biological importance because CCK(58)(ns) binds poorly to the CCK(A) receptor and has only been identified once in intestinal extracts. In this work, a radioimmunoassay specific for the COOH-terminal region of gastrin and CCK (antibody 5135) was used to monitor the purification of CCK molecular forms from canine intestinal extracts. A minor immunoreactive peak was associated with a major absorbance peak during an ion-exchange, HPLC step. Characterization of this minor immunoreactive peak demonstrated that it was CCK(58)(ns). CCK(58)(ns) is 14% as immunoreactive as sulfated CCK(8) [CCK(8)(s)]. Amino acid analysis demonstrated that CCK(58)(ns) was present at 50% the amount of CCK(58)(s). In addition, we found that CCK(58)(ns) does not potently displace an (125)I-labeled CCK(10) analog from the CCK(A) receptor in mouse pancreatic membranes and does not stimulate amylase release from isolated pancreatic acini, or stimulate pancreatic secretion in an anesthetized rat model. By contrast, CCK(58)(ns) does bind to CCK(B) receptors and stimulates gastric acid secretion via this receptor. The presence of CCK(58)(ns) and its ability to selectively stimulate the CCK(B) receptor without stimulation of the CCK(A) receptor suggest that CCK(58)(ns) may have unique physiological properties, especially tissues where the nonsulfated peptide can act as a paracrine or neurocrine agent.

INTRODUCTION AND AIMS:It has been proposed that distinct tertiary structures of the C-terminus of CCK-8 and CCK-58 result in differences in stimulation of pancreatic amylase secretion. Binding of CCK-8 and CCK-58 to CCK-A and CCK-B receptors and stability to enzymatic digestion were used as independent probes for tertiary structure of the C-terminus.METHODOLOGY:Canine CCK-58 was purified from intestinal extracts and CCK-8 was purchased. Their amounts were determined by amino acid analysis. The effect of tertiary structure on receptor binding at CCK-A receptors and CCK-B receptors was evaluated using membrane preparations from mouse pancreas and brain. The influence of C-terminal tertiary structure on stability to enzymatic digestion was evaluated by reacting CCK-8 and CCK-58 with endopeptidase 24:11.RESULTS:CCK-58 was three times more potent than CCK-8 for binding mouse pancreatic membrane CCK-A receptors and equipotent to CCK-8 for binding mouse brain CCK-B receptors. CCK-8 was readily digested by endopeptidase 24:11, whereas CCK-58 was not.CONCLUSIONS:The results strongly support the hypothesis that differences in tertiary structure of the carboxyl terminus of CCK-8 and CCK-58 influence receptor binding and stability to enzymatic digestion.

Sulfated CCK-58 and CCK-8 have identical bioactive C-terminal primary sequences but distinct C-terminal solution structures and different bioactivities. To examine structural differences in greater detail, rat CCK-58 and -8 were synthesized with isotopic enrichment of C-terminal residues with (15)N at alpha-amino nitrogens. Proton and nitrogen chemical shift assignments of peptide solutions were obtained by homo- and heteronuclear NMR methods. These data show that the tertiary structure ensembles of C-terminal CCK-8 and CCK-58 differ significantly. Thus, distinct solution conformations may explain differences in CCK(A) and CCK(B) receptor interactions of large and small molecular forms of CCK.

Background & Aims: Although alcoholism is a major cause of pancreatitis, the pathogenesis of this disorder remains obscure. Failure to produce experimental alcoholic pancreatitis suggests that ethanol may only increase predisposition to pancreatitis. This study sought to develop a model of ethanol pancreatitis by determining if an ethanol diet sensitizes rats to pancreatitis caused by cholecystokinin octapeptide (CCK-8). Methods: Rats were fed intragastrically either control or ethanol diet for 2 or 6 weeks. The animals were then infused for 6 hours-with either saline or CCK-8 at a dose of 3000 pmol . kg(-1) . h(-1), which by itself did not induce pancreatitis. The following parameters were measured: serum amylase and lipase levels, pancreatic weight, inflammatory infiltration, number of apoptotic acinar cells, pancreatic messenger RNA (mRNA) expression of cytokines and chemokines, and nuclear factor (NF)-kappa B activity. Results: All measures of pancreatitis, as well as NF-kappa B activity and mRNA expression for tumor necrosis factor alpha, interleukin 6, monocyte chemotactic protein 1, macrophage inflammatory protein 2, and inducible nitric oxide synthase, were significantly increased only in rats treated with ethanol plus CCK-8. Conclusions: An ethanol diet sensitizes rats to pancreatitis caused by CCK-8. The combined action of ethanol and CCK-8 results in NF-kappa B activation and up-regulation of proinflammatory cytokines and chemokines in the pancreas. These mechanisms may contribute to the development of alcoholic pancreatitis.

The C-terminal bioactive regions of CCK-8 and CCK-58 are identical (DY*MGWMDF-NH2, Y* denotes a sulfated tyrosine residue), but these peptides have different patterns of bioactivity. Specifically, CCK-58 binds more avidly to the CCKA receptor while CCK-8 is more potent for stimulation of amylase secretion from pancreatic acini. We postulate that these seemingly contradictory observations reflect a stable conformational change in CCK-58 that enhances binding, but diminishes activation of second messenger. We used CD and NMR spectra to evaluate postulated structural differences between CCK-8 and the carboxy-terminus of synthetic CCK-58. The CD spectra indicate the presence of turns in CCK-8 but a mixture of helical and random coil structures for CCK-58. Comparisons of partial NMR chemical shift assignments of CCK-58 and full assignments for CCK-8 also indicate differences in the backbone conformations for these residues. The data support the hypothesis that these peptides have different, stable, carboxy-terminal structures that may influence bioactivity. (C) 1999 Academic Press.

TRAVIS E. SOLOMON,1 JOHN H. WALSH,1 LOUIS BUSSJAEGER,2 YUMEI ZONG,1 JAMES W. HAMILTON,2 F. J. HO,1 TERRY D. LEE,3 AND JOSEPH R. REEVE, JR.1 1CURE: Digestive Diseases Research Center, Greater Los Angeles Department of Veterans Affairs Health Care System, Los Angeles 90073; Digestive Diseases Division, University of California, Los Angeles, School of Medicine, Los Angeles, California 90024; 2Kansas City Department of Veterans Affairs Medical Center, Kansas City, Missouri 64128; Department of Biochemistry, Kansas University Medical Center, Kansas City, Kansas 66160; and 3Division of Immunology, Beckman Research Institute, The City of Hope Research Institute, Duarte, California 91010

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The effect of a synthetic analogue of CCK (Thr4,Nle7CCK-9) on growth of SW-1990 human pancreatic cancer was examined in two experimental models. Nude mice bearing SW-1990 pancreatic cancer xenografts were injected with CCK (5, 15, or 25μg/kg) or vehicle twice daily for 20 days. Animals were then sacrificed and tumor volume, weight, protein, and deoxyribonucleic acid (DNA) content were evaluated. SW-1990 cells were grown in vitro and the effects of CCK, secretin, vasoactive intestinal peptide (VIP), and proglumide (a CCK-receptor antagonist) on cell number and DNA synthesis were determined. The highest dose of CCK, 25 μg/kg, significantly increased tumor weight, protein content, and DNA content P<0.005). In vitro, CCK caused significant increases in cell counts of up to 47% at six days and 66% at 12 days compared to control. Graded concentrations of CCK had a biphasic effect on DNA synthesis with significant increases of up to 65% P<0.005). CCK-induced cell proliferation was inhibited by proglumide. Secretin slightly increased cancer cell growth, although not as potently as CCK. VIP or secretin in combination with CCK did not show potentiation. These results indicate that growth of some human pancreatic cancers may be influenced by gastrointestinal peptides, of which CCK is the most potent.

Secretin is present in the intestine of a number of developing species, and plasma secretin levels are elevated in newborn pigs and humans. Secretin stimulates the growth and affects the enzymatic composition of the stomach, small intestine, and pancreas in adult rats. This suggests a possible role for secretin in the rapid postnatal growth of these organs. We investigated this hypothesis by injecting rats subcutaneously with secretin (100 μg/kg) every 12 hr for seven days beginning on postnatal day 3, 6, 13, or 24. Growth parameters (weight, content of protein, DNA) as well as the composition of organ-specific enzymes of the stomach, small intestine, and pancreas were measured. Secretin increased growth parameters of the stomach and small intestine in a similar pattern, and in a quantitatively different fashion from that observed in the pancreas. Secretin's effects were also dependent on postnatal age for all organs studied. These data demonstrate that secretin can influence organ growth and enzyme composition of the stomach, small intestine, and pancreas of developing rats and may be one factor regulating growth and development of these organs.

Gastrin has been shown to stimulate the growth of carcinogenic-induced colon cancer in animals, and some human colon cancers grown in vitroor as xenografts in nude mice. We determined fasting plasma gastrin levels in control subjects and patients with adenomatous polyps or adenocarcinoma of the colon to determine whether abnormal levels occurred in either patient group. Blood samples were obtained from 73 patients undergoing colonoscopy, primarily for evaluation of Hemoccult-positive stools. Fasting plasma gastrin was significantly greater in patients with adenomatous polyps (24.2±5.7 pM, N=25) or colon cancer (84.5±28.5 pM, N=20) than in controls (9.9±0.9 pM, N=28). Elevations were due to gastrin values greater than control mean + 2 sd in nine patients with polyps (19.5–150.2 pM) and eight with cancer (20.7–403.2 pM). None of the patients had identifiable causes (drugs, prior surgery) for elevated gastrin levels. Our results indicate that elevated plasma gastrin occurs in subgroups of patients with adenomatous polyps or adenocarcinoma of the colon. The cause and potential role of elevated gastrin for polyp and tumor growth in these patients is not known.

Authors investigated the inhibitory effect of somatostatin on pancreatic enzyme secretion in vivo and in vitro, in rats. In conscious animals somatostatin strongly inhibited pancreatic amylase secretion. In vitro, however, amylase secretion was not affected by the peptide: it had no affect either on basal secretion, or on stimulated secretion induced via CCK-, or cholinergic receptors. Furthermore, it did not modify nerve-stimulation induced enzyme secretion. The results suggest that the in vivo inhibitory action somatostatin on pancreatic enzyme secretion is an indirect one.

The role of gastrin in regulating pancreatic growth and its interaction with secretin is unclear. We determined the dose-response relationships of pentagastrin for gastric and pancreatic secretion. Doses that stimulated gastric and pancreatic secretion were given every 8 h for 3 or 5 days; trophic responses of pancreas, oxyntic gland area, duodenum, and colon were compared. Interaction of secretin and pentagastrin on pancreatic growth was measured after 5 days of treatment. Pentagastrin was 250 times less potent for stimulation of pancreatic versus gastric secretion. A submaximal dose of pentagastrin for stimulating pancreatic enzyme secretion doubled pancreatic deoxyribonucleic acid synthesis after 3 days. Pentagastrin caused dose-related increases in pancreatic weight after 3 and 5 days. Pentagastrin had no effect on weight, deoxyribonucleic acid synthesis, or deoxyribonucleic acid content of oxyntic gland area, duodenum, or colon. Secretin had additive effects on pentagastrin-induced pancreatic growth. The pancreas appears to be more sensitive than other organs to trophic effects of pentagastrin. Secretin has additive rather than inhibitory effects on pentagastrin-induced pancreatic growth.

We studied effects of graded concentrations of intragastric calcium on acid secretion, residual gastric volume, and serum gastrin and calcium levels. Intragastric titration was performed with solutions of isotonic mannitol or mannitol plus 2.5, 6, 16, 39, and 97 mM CaCl2 in 10 normal and eight duodenal ulcer subjects. Acid secretion was significantly increased above control values by the two highest CaCl2 concentrations in normal subjects and by the three highest CaCl2 concentrations in ulcer subjects. Highest observed acid output to any concentration of CaCl2 was 55% of peak acid output to pentagastrin in normal subjects and 75% in ulcer subjects. Intragastric calcium also released gastrin; correlation between acid secretion and circulating gastrin was weak (r=0.43,P<0.05). Serum calcium was slightly increased but did not correlate with acid secretion. Residual intragastric volume after both control and CaCl2 solutions was much less in ulcer than in normal subjects; calcium did not alter residual volumes.