Molecular forms of cholecystokinin in human plasma during infusion of bombesin

Bombesin is a tetradecapeptide with stimulatory actions on several gastrointestinal functions. Infusion of bombesin (60 pmol/kg. 20 min) into 7 normal subjects induced significant increases in plasma cholecystokinin (CCK) as measured with 2 sequence-specific radioimmunoassays. Employing antibody 1703, specific for carboxyl-terminal CCK-peptides containing at least 14 amino acid residues, plasma CCK concentrations rose from 0.8 +/- 0.2 pmol/l to 9.9 +/- 1.7 pmol/l (p less than 0.005), while using antibody T204, specific for the sulfated tyrosine region of CCK, plasma CCK levels increased from 2.9 +/- 0.5 pmol/l to 12.4 +/- 1.3 pmol/l (p less than 0.005). Plasma samples obtained from 3 subjects during bombesin infusion were fractionated by Sephadex column chromatography. Fractionation revealed 4 molecular forms of CCK: peak I eluted in the void volume and comprised 0-7% of CCK-like immunoreactivity, peak II eluted at 35% and comprised 8-41% of CCK-like immunoreactivity, peak III eluted at 50% and comprised 44-61% of CCK-like immunoreactivity, and peak IV eluted at 75% and comprised 15-27% of CCK-like immunoreactivity. Radioimmunoassay with a carboxyl-terminal CCK-antibody fully cross-reacting with gastrin did not reveal additional molecular forms of CCK. Since both the carboxyl-terminus and the sulfated tyrosine region are required for biological activity of CCK, it is likely that all these molecular forms of CCK possess biological activity.     

Development of cholecystokinin radioimmunoassay using synthetic CCK-10 as immunogen

Using an antiserum generated against synthetic CCK-10, we have developed a radioimmunoassay specific for the carboxyl-terminus of cholecystokinin (CCK). Three rabbits were immunized with synthetic sulfated carboxy-terminal CCK decapeptide (CCK-10) conjugated to keyhole limpet hemocyanin. Using 125I-CCK-39 prepared by the Iodogen method as a tracer, we found that all immunized rabbits produced antibodies against the conjugate. Antiserum R016 had the highest titer (1:225,000 after four immunizations) and was studied most extensively. R016 recognizes all molecular forms of CCK, including unsulfated and oxidized forms, but has negligible cross-reactivity with gastrin and other peptides. Using CCK-8 as a standard, the assay has a minimum detection limit of 0.5 pM and an ED50 of 11.5 pM. Serial dilutions of water/acid extracts of canine intestine were parallel to serial dilutions of sulfated CCK-8, CCK-33 and CCK-39. The assay was used to measure CCK concentrations in canine plasma after C18 Sep-Pak extraction; the concentration of immunoreactive CCK increased from a basal value of 7.8 +/- 1.0 to 9.5 +/- 1.2 and 11.1 +/- 1.2 pM 30 and 60 min postprandially (P less than 0.05 by paired analysis). This sensitive and uniquely specific CCK radioimmunoassay should be useful in characterizing several aspects of CCK physiology and the method for generating CCK antisera should be of value to other investigators.     

Plasma cholecystokinin-octapeptide like immunoreactivity in patients with hepatic cirrhosis

Molecular forms of cholecystokinin (CCK) in the peripheral circulation were studied in normal subjects and cirrhotic patients. Fractionation of plasma extract collected 20 min after intraduodenal infusion of fat revealed four major peaks by Sephadex G-50 column chromatography in normal subjects. Peak I eluted at a position similar to CCK-33, peaks II and III eluted between CCK-33 and CCK-14, and peak IV eluted between CCK-14 and CCK-8. In cirrhotic patients, there was a prominent peak (peak V) eluted at a position similar to CCK-8, in addition to those four peaks. These findings are consistent with the previous observations of hepatic elimination of CCK-8, and suggest that smaller forms of CCK similar in size to CCK-8 are not major forms of CCK in plasma in normal subjects but circulate substantially in cirrhotic patients.     

Immunoreactive cholecystokinin in human and rat plasma: correlation of pancreatic secretion in response to CCK

Immunoreactive cholecystokinin (CCK) levels in human and rat plasma are described using a radioimmunoassay specific for the biologically active sulfated end of CCK. This assay detected significant changes in plasma cholecystokinin levels during intrajejunal administration of amino acids and intravenous infusions of CCK-8 which were followed by increased pancreatic secretion. In humans, the concentration (pg/ml) of plasma cholecystokinin increased from 10.8 to 18.9 following intrajejunal amino acid instillation and from 15.4 to 31.1 during CCK infusion, while pancreatic trypsin secretion increased more than 15 fold. Ingestion of a test meal also caused a rapid and significant elevation (P less than 0.05) in both plasma CCK (14.5-21.7 pg/ml) and gastrin (50-160 pg/ml) levels. In the rat, an injection of 46 ng of CCK-8 produced a 300% increase in immunoreactive plasma CCK levels (2 min) and caused peak pancreatic protein secretion within 5 min; 4 fold lower doses (11.5 ng) elevated plasma CCK by 38% and pancreatic protein secretion to a small but significant extent. The ability of this assay to detect various forms of sulfated CCK in human plasma was also determined. Following gel chromatography on Sephadex G-50, at least three different immunoreactive peaks were found in plasma from fasted subjects and after intrajejunal amino acid stimulation. While the lower molecular weight CCK peptides (CCK-8 and CCK-12) were detected in plasma from both fasted and stimulated subjects, the larger form (CCK-33) was only present in measurable concentrations after amino acid infusion. The simultaneous measurement of increased plasma CCK levels and pancreatic secretion and the changes in the distribution of CCK peptides following amino acid infusion provides strong support that this assay detects physiologically relevant changes in biologically active CCK peptides.     

The molecular nature of vascularly released cholecystokinin from the isolated perfused porcine duodenum

Using sequence-specific radioimmunoassays, the quantities and molecular nature of cholecystokinin (CCK) have been determined in extracts of porcine duodenal mucosa and in the vascular perfusate from the isolated porcine duodenum. The basal concentration of CCK in the perfusate was 84 pM equiv. CCK-8 (mean; range: 32–173 pM, n = 5). After intraluminal stimulation with amino acids, acidified fat emulsions and hydrochloric acid, the concentrations increased 2–5-fold. Both in the basal and stimulated state the concentrations of the related hormone, gastrin, were below 5 pM equiv. gastrin-17. CCK in the perfusate was concentrated by affinitychromatography using antibodies directed against the bioactive C-terminus. Subsequent gel chromatography revealed a form with a size like or slightly larger than the C-terminal dodecapeptide (CCK-12), a predominant form resembling the C-terminal octapeptide (CCK-8), and a form resembling the C-terminal tetrapeptide (CCK-4). The duodenal mucosa contained in addition CCK-33, -39 and CCK-peptides with further N-terminal extensions. The results suggest that small CCK peptides are the principal circulating forms, while CCK-33 and larger forms are biosynthetic precursors.     

Isolation and characterization of biologically active and inactive cholecystokinin-octapeptides from human brain

Cholecystokinin-like immunoreactivity (CCK-LI) in 0.9 kg human brain was extracted by 2% trifluoroacetic acid at 4 degrees C. Sephadex G50 gel filtration of crude extract revealed one main molecular form of CCK, detected by a carboxy-terminal antibody (5135), that eluted in the position of CCK8. When the CCK-LI in the extract was purified by affinity chromatography using another carboxyl-terminal CCK antibody followed by several steps of reverse phase high pressure liquid chromatography (HPLC), a component was isolated that was found by sequence analysis to be identical to the carboxyl-terminal CCK-octapeptide of porcine CCK33, isolated from intestinal mucosa, and to CCK-octapeptide, isolated from sheep brain. This component possessed comparable biological potencies to synthetic sulfated CCK8 in eliciting amylase release and in competitively displacing radioiodinated CCK33 from isolated mouse pancreatic acini. Furthermore, it exhibited a similar binding characteristic to CCK8 in binding to specific receptors on mouse brain cortical particulate preparations. On high pressure liquid chromatography another minor, earlier eluting immunoreactive peak was observed, which had the same amino acid composition and sequence as CCK8. These findings suggested that this material was oxidized CCK8. This earlier eluting component, exhibiting CCK8-like immunoreactivity, did not induce amylase release from acini and had no or minimal effect in inhibiting tracer CCK33 binding to receptors on isolated acini or on mouse brain cortical particulate preparations at the concentrations tested.     

Physiological plasma concentrations of cholecystokinin stimulate pancreatic enzyme secretion and gallbladder contraction in man

The present study was undertaken to determine whether infusion of cholecystokinin (CCK) to plasma concentrations comparable to those found after a meal stimulates pancreatic enzyme secretion and gallbladder contraction. Plasma CCK concentrations were measured by radioimmunoassay using antibody T204, which binds to all carboxyl-terminal CCK-peptides containing the sulfated tyrosine region. Ingestion of a standardized test meal in 7 normal subjects induced significant increases in plasma CCK from 2.0 +/- 0.2 pmol/l to levels between 4.6 +/- 0.6 and 7.3 +/- 1.0 pmol/l (p less than 0.05-p less than 0.0005). Infusion of 2.5 pmol/kg X h CCK 33 resulted in significant increases in plasma CCK from 2.0 +/- 0.2 to 3.9 +/- 0.3 pmol/l (p less than 0.0005). This infusion of CCK induced significant increases in trypsin secretion from 0.5 +/- 0.1 to 1.4 +/- 0.2 KU/15 min (p less than 0.005) and in bilirubin output from 1.6 +/- 0.7 to 30.3 +/- 8.0 mumol/15 min (p less than 0.05). It is concluded that physiological plasma concentrations of CCK stimulate pancreatic enzyme secretion and gallbladder contraction in man.     

Evidence against autocrine feedback regulation of cholecystokinin secretion in man.

To determine whether exogenous cholecystokinin (CCK) inhibits endogenous CCK release, cholecystokinin-8S (CCK-8S) was infused intravenously during continuous intraduodenal stimulation of endogenous CCK by a meal. CCK was measured in plasma by 2 region-specific radioimmunoassays employing antibodies T204 and 1703. AB T204 binds to carboxy-terminal CCK peptides containing the sulphated tyrosyl region, including CCK-8S, and AB 1703 to carboxy-terminal CCK peptides containing at least 14 amino acid residues. Meal-stimulated plasma CCK concentrations remained elevated during the entire infusion period. CCK-8S infusion further increased meal-stimulated plasma CCK concentrations, when measured with AB T204, while meal-stimulated plasma CCK concentrations were not suppressed by CCK-8S infusion, when measured with AB 1703. It is concluded that meal-stimulated endogenous CCK release is not affected by the effects of intravenously administered CCK-8S. These data suggest that autocrine feedback regulation of CCK release is not operative in man.     

Receptor binding of cholecystokinin analogues in isolated rat pancreatic acini

The receptor binding of CCK analogues was determined in terms of the inhibition of [125I]CCK binding in isolated rat pancreatic acini. The inhibition curve produced by CCK-8 showed the same feature as that produced by synthetic human CCK-33. The relative potency values of CCK analogues to half-maximally inhibit specific CCK binding were calculated; CCK-8 was equal to human CCK-33, 3-fold stronger than natural porcine CCK-33 and 39, and 700-fold stronger than the unsulphated form of synthetic human CCK-33. Our data suggest that CCK-33, one of the longer molecular forms of CCK, is as important as CCK-8 in the mechanism of physiological actions of CCK.     

Characterization of canine intestinal cholecystokinin-58 lacking its carboxyl-terminal nonapeptide. Evidence for similar post-translational processing in brain and gut.

An antibody raised against a synthetic cholecystokinin (CCK) analog, (1-27)-(CCK)-33, corresponding to the midregion of CCK-58, detected immunoreactivity in intestinal extracts which eluted between the positions of CCK-33/39 and CCK-58 on high performance liquid chromatography. This peak, lacking carboxyl-terminal cholecystokinin immunoreactivity, was purified by reverse phase and cation-exchange chromatographies. Amino acid, mass spectral, and microsequence analysis established that it was the amino-terminal desnonapeptide fragment of cholecystokinin-58, (1-49)-CCK-58. It was demonstrated further that CCK-58 has less biological activity than CCK-8, suggesting that the amino terminus either sterically hindered the ability of CCK-58 to exert its biological activity or that its amino terminus acted at another site to inhibit release of amylase from rat pancreatic acini. The desnonapeptide of CCK-58 by itself had no biological activity, nor did it affect CCK-8-stimulated amylase release from isolated rat pancreatic acini, suggesting that the amino terminus shields the carboxyl terminus from expressing its biological activity. Its presence in intestine suggests that it is released into the circulation where it could be detected by midregion antibodies. The presence of high proportions of (1-49)-CCK-58 indicates that most CCK-8 is directly derived from CCK-58. Its occurrence in brain and intestine indicates similar processing for procholecystokinin in both tissues.     

Effects of cholecystokinin octapeptide on the pancreatic exocrine secretion in the pig

In conscious pigs, intravenous infusion of serial doses of cholecystokinin octapeptide (CCK8; 2.9-232.3 pmol.kg-1.min-1) upon a background of secretin resulted in a linear increase of plasma CCK-like immunoreactivity (CCK-LI) concentration and evoked a dose-related increase of pancreatic volume and bicarbonate and protein outputs. The threshold plasma CCK-LI concentration for significant pancreatic response was 103.8 +/- 10.2 pM using a CCK8 dose of 8.8 pmol.kg-1.min-1. The maximum pancreatic response was observed for a plasma CCK-LI level of 498.0 +/- 15.3 pM using 77.2 pmol CCK8.kg-1.min-1. In anesthetized pigs, the threshold plasma CCK-LI concentration for pancreatic response was 1500 pM (actual CCK8 dose of 60.3 pmol.kg-1.min-1). The physiological relevance of this finding was assessed by comparing the food-induced increase of pancreatic secretion with that of plasma CCK-LI. Food ingestion was followed by a sharp pancreatic response and by a progressive increase of plasma CCK-LI to a peak increment of about 15 pM. Gel chromatography of portal and peripheral plasma from fed animals revealed three major peaks in the volumes of CCK33/39 and CCK8, and in a volume intermediate between CCK33/39 and CCK8. An additional minor component eluted ahead of CCK33/39. CCK8, which is one of the CCK components released after food intake, appears to be a fairly weak pancreatic stimulant in pigs.     

Molecular heterogeneity of canine cholecystokinin in portal and peripheral plasma

The release of molecular forms of cholecystokinin (CCK) into the portal and peripheral blood in response to an intraduodenal perfusion of sodium oleate (9 mmol X h-1) was studied in six conscious dogs with chronic portal vein catheters. Immunoreactive CCK as concentrated from 20 ml plasma by C18 SEP PAK cartridges and the pattern of molecular forms of CCK were studied by G50 gel filtration. CCK-like immunoreactivity (CCK-LI) was measured in the column eluates with antibody 5135, which measures gastrin and CCK equally and requires the intact carboxyl-terminus for full recognition. Gastrin was measured specifically with antibody 1611. Intraduodenal perfusion with oleate did not alter basal gastrin release. Release of CCK-LI by intraduodenal oleate was calculated by the increments of the integrated CCK-LI peaks over basal. Total CCK-like immunoreactivity (CCK-LI), calculated by integration of all CCK-LI peaks in gel filtration eluates, increased over basal by 12 fmol/ml in the portal and by 6 fmol/ml in the peripheral plasma after intraduodenal perfusion with sodium oleate. The main molecular forms eluted on gel filtration in positions of CCK33,39 and of CCK8. The pattern of CCK in the peripheral plasma was similar to that in the portal plasma except that in the peripheral plasma large molecular forms were more abundant than small forms. This finding was confirmed when CCK39 and CCK8 were infused either into the portal vein or into the peripheral vein and peripheral plasma CCK levels were measured. Elimination of CCK8 after portal vein infusion compared to peripheral vein infusion was about 3 times higher than that of CCK39. The abundance of large molecular forms of CCK in the circulating blood which are similar in potency to small forms, underlines their role in the physiology of CCK.     

An amino-terminal fragment of cholecystokinin-58 is present in the gut: evidence for a similar processing site of procholecystokinin in canine gut and brain

Radioimmunoassays using antibodies specific for the carboxyl terminus of cholecystokinin (CCK) and the midportion of CCK-58 (raised against synthetic canine CCK-33-(1-27] revealed the existence of a CCK fragment in canine gut and brain extracts which lacks the biologically active carboxyl terminal immunoreactivity. This material eluted on Sephadex G-50 gel permeation chromatography in the region of CCK-58, on high-pressure liquid chromatography (HPLC) after CCK-39 and before CCK-58, and on cation-exchange FPLC it eluted after CCK-58. The immunoreactive pattern, the ratio of absorbance at 280-220 nm and the chromatographic elution positions suggest that this large CCK-like molecule represents an amino-terminal fragment of CCK-58. This fragment is present in canine gut and brain. Therefore, a similar processing site of procholecystokinin is suggested in both tissues.     

Rat immunoreactive cholecystokinin (CCK): characterization using two chromatographic techniques

Acid and neutral extracts of rat cerebral cortex and upper small intestine were prepared and the endogenous concentrations of cholecystokinin-like immunoreactivity (CCK-LI) measured by three new CCK-specific radioimmunoassays. The characterization of the immunoreactive CCK molecular forms was undertaken using gel permeation chromatography in the presence of 6 M urea to minimise problems relating to peptide adsorption or aggregation. Reverse-phase high-performance liquid chromatography (HPLC) was also performed on the rat tissue extracts. Rat cortex contained 268 +/- 12 pmol/g CCK-LI, and over 90% resembled the sulphated CCK-8, which was preferentially extracted at neutral pH. In contrast, the rat upper small intestine (97 +/- 8 pmol/g of CCK-LI) contained less than 20% CCK-8, the majority of immunoreactive CCK being of larger molecular size and being preferentially extracted at acid pH. In the small intestine the predominant molecular form(s) was intermediate in size between CCK-33 and CCK-8. Large amounts of CCK-33 and of a molecular form larger than CCK-33 were also detected. It is concluded that post-translational cleavage of CCK differs in rat brain and gut.     

Cholecystokinin-58 is the major molecular form in man, dog and cat but not in pig, beef and rat intestine

Cholecystokinin (CCK)-58 was found to be the most abundant form in upper small intestinal mucosa of man, dog and cat. However, in pig, beef and rat upper small intestinal mucosa CCK-33/39 and smaller CCK-forms were dominant. The differences in the distribution of the molecular forms of cholecystokinin between these species presumably reflects altered posttranslational processing of procholecystokinin. This may be caused by the different feeding habits of the investigated species. The different forms of cholecystokinin were distributed over the entire length of the mucosa in canine small intestine. The total amount of CCK decreased from the duodenal mucosa towards the colon. In the canine duodenal mucosa, CCK-58 accounted for 85% of the total CCK-like immunoreactivity. The relative amounts of small forms of CCK increased towards the distal jejunum.     

Cholecystokinin-58 is the major circulating form of cholecystokinin in canine blood

Cholecystokinin-58 (CCK-58) is the largest and most abundant, biologically active form of cholecystokinin in canine intestinal mucosa. Despite the high amounts in mucosa, CCK-58 has not been detected in significant amounts in the circulation. The release of CCK-58 into the peripheral blood in response to an intraduodenal perfusion of sodium oleate (9.0 mmol h-1) was studied in seven conscious dogs. Plasma (50 ml) was obtained before and after endogenous stimulation by a newly developed method that prevents in vitro degradation of large cholecystokinins. The relative abundance of immunoreactive forms of CCK was studied by high pressure liquid chromatography (HPLC) which separated the gastrin and CCK forms. Column eluates were measured with an antibody which recognizes the intact carboxyl terminus of both gastrin and CCK. Cholecystokinin immunoreactivity increased over basal in plasma by 7 fmol/ml after intraduodenal perfusion with sodium oleate. The most abundant form of stimulated cholecystokinin immunoreactivity eluted on HPLC in the position of CCK-58 (63% of total immunoreactivity found). Since CCK-58 is biologically active and is the most abundant circulating form, it should play an important role in the physiology of cholecystokinin.     

Canine vagus nerve stores cholecystokinin-58 and -8 but releases only cholecystokinin-8 upon electrical vagal stimulation

Cholecystokinin-58 has been shown to be the major form of cholecystokinin (CCK) released to the circulation upon lumenal stimulation of the small intestine in humans and dogs. In anesthetized dogs, electrical vagal stimulation evokes pancreatic exocrine secretion that is in part mediated through the release of CCK. We studied the molecular form of CCK stored in canine vagus nerves and that released into circulation upon electrical vagal stimulation. Gel filtration and radioimmunoassay of the water and acid extracts of canine vagus nerves indicated CCK-8 (35%) and CCK-58 (65%) as the major molecular forms in the vagus nerve. Both forms of CCK isolated from the vagal extracts were equally bioactive as the standard CCK-8 and CCK-58, respectively, in stimulation of amylase release from isolated rat pancreatic acini. Analysis of plasma collected after electrical vagal stimulation indicated that CCK-8 is the only form released into the circulation. The release of CCK-8 upon electrical vagal stimulation was not affected by application of lidocaine to the upper small intestinal mucosa, suggesting that it was released from vagal nerve terminals.     

Oxidation/reduction of methionine residues in CCK: a study by radioimmunoassay and isocratic reverse phase high pressure liquid chromatography

The study was undertaken to investigate the oxidation and reduction of cholecystokinin (CCK) both as pure standards and as endogenous porcine peptides. Furthermore an attempt was made to prevent oxidation of the endogenous porcine peptides in the extraction procedure. CCK-8 and CCK-33 standards were always oxidized in weak solutions, CCK-8 varying from 26% to 67% oxidized and CCK-33 from 18% to 70%. Similarly, tissue extracts of porcine brain and duodenum contained oxidized forms of the peptide. CCK standards were readily oxidized in the presence of hydrogen peroxide. Oxidized CCK-8 standard and CCK-8 in porcine brain was 90% reduced and oxidized CCK-33 standard and in duodenal extracts was reduced by 70% by a 40 hour incubation with 0.725 mol/l dithiothreitol at 37 degrees C. Extraction of CCK peptides in the presence of 65 mmol/l dithiothreitol resulted in almost complete prevention of oxidation with over 95% of the peptides being obtained in the reduced state. This additive is therefore recommended for all tissue quantitation studies.     

Cleavage-site mutagenesis alters post-translation processing of Pro-CCK in AtT-20 cells

Cholecystokinin (CCK) is expressed in the central and peripheral nervous systems and functions as a neurotransmitter and neuroendocrine hormone. The in vivo forms of CCK include CCK-83, -58, -39, -33, -22, -12, and -8. Tissues in the periphery produce the larger forms of CCK, such as CCK-58, whereas the brain primarily produces CCK-8. The different biologically active forms of CCK observed in vivo may result from cell-specific differences in endoproteolytic cleavage during post-translational processing. Evidence suggests that cleavages of pro-CCK occur in a specific sequential order. To further delineate the progression of cleavages during pro-CCK maturation, mutagenesis was used to disrupt putative mono- and dibasic cleavage sites. AtT-20 cells transfected with wild-type rat prepro-CCK secret CCK-22 and -8. Mutagenesis of the cleavage sites of pro-CCK had profound effects on the products that were produced. Substitution of basic cleavage sites with nonbasic amino acids inhibits cleavage and leads to the secretion of pathway intermediates such as CCK-83, -33, and -12. These results suggest that CCK-58 is cleaved to both CCK-33 and -22. Furthermore, CCK-8 and -12 are likely derived from cleavage of CCK-33 but not CCK-22. Alanine substitution at the same site completely blocked production of amidated products, whereas serine substitution did not. The cleavages observed at nonbasic residues in this study may represent the activity of enzymes other than PC1 and carboxypeptidase E, such as the enzyme SKI-1. A model for the progression of pro-CCK processing in AtT-20 cells is proposed. The findings in this study further supports the hypothesis that pro-CCK undergoes parallel pathways of proteolytic cleavages.     

Chemical characterization of rat cholecystokinin-58

Cholecystokinin-58 (CCK-58) was purified from rat intestines using an extraction method that yields large amounts of this peptide. Greater than 30% of total CCK immunoreactivity eluted before CCK-39 upon gel permeation chromatography (Sephadex G-50) if extracts were loaded onto Sep Pak cartridges before freezing. If the extracts were frozen and stored at −70°C for six weeks, only 20% of the material eluted in this region and total immunoreactivity was reduced by 50%, suggesting that proteases were active under these storage conditions. This early eluting peak was purified by reverse phase and ion-exchange HPLC to a single absorbance peak. Microsequence analysis of this peak detected AVLRPDSEP which is the amino terminus of rat CCK-58 predicted from the rat preprocholecystokinin cDNA. Because degradation of CCK-58 occurred in these extracts, it is possible that CCK-58 is the predominant molecule form in the rat small intestine.