Evidence that CCK-58 has structure that influences its biological activity.

Many biologically active peptides exist in multiple molecular forms, but the functional significance of regions outside the region of bioactivity is unknown. The biological and immunological data presented in this study indicate that cholecystokinin-58 (CCK-58), unlike other forms of cholecystokinin, has structure that influences its bioactivity. CCK-58 was purified from acid extracts of canine intestinal mucosa until a single absorbance peak was obtained during reverse-phase chromatography. Amino acid analysis precisely determined the peptide concentrations of purified CCK-58 and synthetic CCK-8. Our hypothesis was that if the amino terminus of CCK-58 influences its bioactivity then its activity would be modified when this region was removed from the peptide. To evaluate the importance of the amino terminus of CCK-58 to influence its biological activity, the abilities of CCK-58 and CCK-8 to release amylase from pancreatic acini were compared before and after tryptic digestion. Tryptic digestion of CCK-58 decreased the half-maximal stimulation (EC50) for amylase release from 96 to 28 pM. The EC50 for digested CCK-58 was similar to that for CCK-8 (17 pM). These results suggest that CCK-58 has a structure that shields its bioactive carboxyl terminus. This is further supported by the finding that carboxyl fragments generated from CCK-58 by trypsin or by partial acid hydrolysis were greater than twofold more immunoreactive than the intact CCK-58. The diminished activity of CCK-58 SK shields the carboxyl terminus, which is important to its biological and immunological activities.

Determination of various molecular forms of cholecystokinin from canine mucosa by radioimmunoassay and bioassay.

Bioassays using amylase release from isolated pancreatic acini measure only cholecystokinin (CCK) forms with an intact carboxyl terminus ending with phenylalanine amide, but it cannot be excluded that peptides not structurally related to CCK are also responsible for CCK-like bioactivity. CCK exists in several molecular forms in intestinal mucosa which are released into the circulating blood. We studied the molecular forms of CCK in canine intestinal extracts after separation by high performance liquid chromatography by bioassay and compared them with those detected by radioimmunoassay. For the radioimmunoassay, an antibody was used which needs the carboxyl terminal phenylalanine amide for recognition. Three immunoreactive peaks were reproducibly seen in HPLC eluates which eluted in the regions of synthetic CCK-8, purified porcine CCK-33-39 (which co-elute using this gradient) and purified canine CCK-58. All these peaks were bioactive for amylase release from isolated pancreatic acini. No further bioactive peaks were detected in the HPLC eluates. When an antibody was used which recognizes the midregion of CCK-58, an additional peak was detected which eluted between CCK-33-39 and CCK-58. This form presumably represents an amino terminal fragment of CCK lacking the carboxyl terminus. It can be concluded that bioassays of CCK measure only CCK bioactivity in intestinal mucosal extracts, whereas radioimmunoassays may detect biologically inactive forms depending on the antibody recognition site.

Influence of treatment with pancreatic extracts on pancreatic enzyme secretion.

We have evaluated the effects of porcine pancreatic extracts on human pancreatic secretion. Ten male volunteers were intubated with a 4-lumen jejunal tube to collect gastric and duodenal secretions separately via the first and third tube, to infuse PEG 4000 distal the pylorus via the second tube and to apply porcine pancreatic extracts via the fourth tube distal the ligament of Treitz. Pancreatic extracts were given four times at 40 minute intervals; the first two as active enzymes and subsequently as heat denatured proportions. Secretin was continuously infused intravenously (0.5 E/kg bw/h) to achieve minimal pancreatic flow. Lipase, amylase, trypsin, chymotrypsin, volume, and bicarbonate were measured in duodenal contents in eight pooled 15 minute fractions. Three subjects who received HEPES-Ringer buffer instead of pancreatic enzymes served as controls. Plasma cholecystokinin (CCK) was measured using a sensitive bioassay. Both active and heat denatured pancreatic extracts caused a small but significant increase in amylase and chymotrypsin secretion. Basal plasma CCK values were 0.85 (0.05) pM. After intrajejunal instillation of either active or heat denatured pancreatic extracts plasma CCK rose to 3.25 (0.30) pM and to 3.28 (0.36) pM respectively. In a second group of five volunteers, plasma CCK concentrations were measured after a test meal. On day 1, volunteers received a liquid fat and protein rich meal and on day 2, the same test meal containing porcine pancreatic extracts. In both cases, a similar increase in plasma CCK was observed. We conclude that therapy with pancreatic extracts stimulate pancreatic enzyme secretion. This may be mediated through release of CCK.

[Plasma cholecystokinin level in chronic pancreatitis]. / Plasma-Cholezystokininspiegel bei chronischer Pankreatitis.

Secretion of pancreatic enzymes is inhibited in rats by the presence of intraduodenal proteases via inhibition of CCK release. The existence of a similar feedback mechanism in man is discussed controversially. Thus, in chronic pancreatitis (cP), which leads to a decrease of digestive enzyme secretion, increases in plasma CCK may be postulated. However, food induced CCK release may be impaired in cP due to maldigestion. We studied, therefore, the influence of food with or without addition of pancreatic extracts on plasma CCK in 16 male patients with longstanding cP. Plasma CCK was measured by bioassay using pancreatic rat acini prepared by collagenase digestion. Plasma samples were processed through SEP-PAK cartridges and assayed for CCK-like activity by comparing the bioactivity of samples with those of standard curves of CCK8. Plasma CCK was measured in 20 healthy controls and in cP prior and 7.5, 15, 30, 45, 60, and 90 min after the application of a test meal made out of milk, cream, eggs, and cacao. In addition CCK was measured in 10 of the same patients with cP on a separate day but with the addition of pancreatic extracts to the test meal. Basal plasma CCK levels were similar in both groups (control: 1.3 +/- 0.2 vs. cP: 1.5 +/- 0.3 pMol/l). Both groups showed a similar steep increase of postprandial CCK with maximal values seen between 7.5 and 30 min (control: 4.6 +/- 0.6 vs cP: 4.8 +/- 1.3 pMol/l). The addition of pancreatic extracts to the liquid meal in cP caused a statistically significant slight increase in plasma CCK.(ABSTRACT TRUNCATED AT 250 WORDS)

Influence of food on plasma cholecystokinin and gastrin in patients with partial gastric resections and Roux-en-Y anastomosis.

Cholecystokinin (CCK) is a polypeptid released postprandially by the upper intestinal mucosa. There are several biological active forms of CCK. Radioimmunological measurements of CCK may not detect all biological active forms or may have the disadvantage of crossreacting with gastrin. In the following we describe a modification of a bioassay for CCK which was first developed by Liddle et al. (J Clin Invest 1985). By means of this bioassay pre- and postprandial plasma CCK-levels of healthy male volunteers are compared with CCK-levels of patients with partial gastric resections and excluded duodenum. Both groups showed similar basal CCK-values (about 1 pM) and a food induced increase of this hormone by reaching maximal values after 15 to 30 min (control: 4.30 +/- 0.65 vs. operated: 13.37 +/- 2.83 pM). Patients with gastric resections, however, had about three times more CCK released over the 60 min time period studied as compared to controls. Thus exclusion of the duodenum, the supposed main place of CCK production, does not cause a lower but rather higher increase of postprandial CCK release.