Relative bioactivities of cholecystokinins-8 and -33 on rat pancreatic acini

The relative potencies of cholecystokinin (CCK-33) and its carboxyl terminal octapeptide (CCK-8) for stimulation of amylase release from rat pancreatic acini was measured. Porcine CCK-33 and synthetic CCK-8 were initially subjected to high pressure liquid chromatography to assess purity. Concentrations of each peptide were determined by amino acid analysis. The relative immunoreactivities of CCK-33 and CCK-8 were compared using an antibody that recognizes the common carboxyl terminus of these forms. This antibody bound CCK-8 and CCK-33 with nearly equal affinity. The relative potencies of CCK-33 and CCK-8 were then measured by comparing their abilities to stimulate amylase release from isolated rat pancreatic acini. Statistical analysis of the relative potencies of the two hormones indicated that CCK-8 was 36% more potent than CCK-33 in this assay system. These data suggest that differences in biological activities between large and small forms of CCK are not as great as previously reported.     

Correlation between phospholipid breakdown, intracellular calcium mobilization and enzyme secretion in rat pancreatic acini treated with Boc-[Nle28, Nle31]-CCK-7 and JMV180, two cholecystokinin analogues.

In this work in vitro pharmacological profiles of two analogues of the C-terminal heptapeptide of cholecystokinin (CCK) were evaluated. The analogue Boc-[Nle28, Nle31]-CCK-7, a stable analogue of CCK-8, has the same activity profile as CCK-8, and was found to be very potent in stimulating amylase secretion, phospholipid breakdown and [Ca2+]i mobilization from rat pancreatic acini. It can be used as a probe for studying CCK-actions. The CCK-analogue Boc-Tyr(SO3H)-Nle-Gly-Trp-Nle-Asp-2-phenylethylester, (JMV180), which stimulates amylase secretion without inhibition at supramaximal concentrations, has different effects on phospholipid hydrolysis and [Ca2+]i mobilization, compared to CCK-8 and Boc-[Nle28, Nle31]-CCK-7. Compound JMV180 was unable to significantly promote phospholipid breakdown, and was only 50%-60% as efficacious as Boc-[Nle28, Nle31]-CCK-7 in promoting [Ca2+]i mobilization. These findings suggest that low affinity CCK-receptors might be responsible for the supra-maximal inhibition of amylase secretion, and are correlated with phospholipid breakdown and maximal [Ca2+]i mobilization.     

Characterization of interactions between CCK-33 and CCK receptors in isolated dispersed pancreatic acini.

In isolated dispersed pancreatic acini, we have characterized the interactions between cholecystokinin (CCK) and CCK receptors by simultaneously measuring CCK-33 immunoreactivity and CCK bioactivity. Incubation of acinar cells with CCK-33 at cell density of 0.2-0.3 mg acinar protein per ml resulted in stimulation of amylase release concomitant with significant and time-dependent decrease of the immunoreactive CCK. With L-364,718 (0.1 microM), a specific CCK receptor antagonist, immunoreactive CCK levels in the media were not significantly altered during incubation; however, CCK-stimulated amylase release was almost completely abolished (94% inhibition). Vasoactive intestinal peptide (1 nM) significantly potentiated CCK stimulated amylase release without affecting immunoreactive CCK in the media. Insulin (167 nM) did not affect the CCK stimulated amylase release or immunoreactive CCK in the media. Incubation of acinar cells with CCK-33 at 4 degrees C did not affect the levels of immunoreactive CCK; however, a significant change in levels of immunoreactive CCK were found at 37 degrees C at 90 min. Incubation of cell free medium with CCK-33 in the presence or absence of secreted enzymes revealed no changes in CCK immunoreactivity in the medium at 90 min. Addition of bacitracin in the incubation media did not affect the CCK immunoreactivity or bioactivity. These findings indicate that in isolated rat pancreatic acini, CCK-33 stimulates amylase release through a receptor that is specifically blocked by L-364,718. Specificity of the interactions of CCK-33 with acinar cells in the media appears to be receptor-mediated and time- and temperature-dependent.     

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.     

Cholecystokinin receptor occupation and cholecystokinin-induced calcium mobilization in the early phase in rat pancreatic acini.

We examined receptor occupation, calcium mobilization and amylase release for cholecystokinin octapeptide (CCK-8) within a 3-min incubation period at 37 degrees C using dispersed acini from rat pancreas. Analysis of competitive binding inhibition data obtained after a 3-min incubation revealed the presence of only a single class of CCK receptors, while two classes of CCK receptor, i.e., high-affinity and low-affinity CCK receptors, were detected when binding reached a steady-state after a 60-min incubation. The IC50 of CCK receptors calculated from the 3-min binding data was 19.0 +/- 0.5 nM (mean +/- S.D.), close to the Kd of the low-affinity CCK receptors determined by equilibrium binding studies. Exposure of fura-2-loaded acini to 10-1000 pM CCK-8 caused an immediate and dose-dependent increase in [Ca2+]i followed by a gradual decrease in [Ca2+]i. The CCK-stimulated amylase release after 3 min of incubation was biphasic; amylase release increased over the dose range of 3-300 pM CCK-8, peaked at 300 pM CCK-8 and decreased with supramaximal concentrations of CCK-8. Our data suggest that occupation of the low-affinity, but not the high-affinity, CCK receptors is more directly associated with calcium mobilization and subsequent stimulation of amylase release in rat pancreatic acini.     

Relationship of CCK/gastrin receptor binding to amylase release in dog pancreatic acini

Effects of synthetic peptides belonging to the CCK/gastrin family (CCK-39, CCK-8, G/CCK-4, G-17ns) on amylase release in dog pancreatic acini have been measured and correlated with binding of three radio-labelled CCK/gastrin peptides: ¹²⁵I-BH-(Thr,Nle)-CCK-9, ¹²⁵I-BH-(2–17)G-17ns and ¹²⁵I-BH-G/CCK-4 prepared by conjugation of the peptides to iodinated Bolton-Hunter reagent and purified by reverse-phase-HPLC. All the CCK/gastrin peptides produced the same maximal amylase release response. Half-maximal responses (D₅₀) were obtained with 2 · 10^?10 M CCK-8; 6 · 10^?10 M CCK-39; 10^?7 M G.17 ns and 2 · 10^?6 M G/CCK-4. Dose-response curves for G-17 ns and G/CCK-4 were similar in configuration but not parallel with those for CCK-8 and CCK-39.Binding studies with ¹²⁵I-BH(Thr,Nle)-CCK-9 demonstrated the presence of specific CCK receptors on dog pancreatic acini. There was a good correlation between receptor occupancy by CCK-8 and CCK-39 and amylase stimulation since maximal amylase stimulation was achieved when 40–50% of high affinity receptors were occupied. In contrast, a saturation of these receptors was required for maximal stimulation by G-17 ns and G/CCK-4 suggesting the existence of a fraction of receptors that can be occupied by G-17 ns and G/CCK-4 without stimulation of amylase release. Binding studies with labelled (2–17)-G-17 ns and G/CCK-4 confirmed the presence of high affinity sites for G-17 ns and G/CCK-4. These sites were not related to amylase release.This study points out a possible species specificity of biological action of gastrin/CCK peptides on pancreatic exocrine secretion in higher mammals.     

A synthetic peptide derivative that is a cholecystokinin receptor antagonist

So far, there are no known peptidic effective receptor antagonists of both peripheral and central effects of cholecystokinin (CCK). Here, we describe a synthetic peptide derivative of CCK, t-butyloxycarbonyl-Tyr(SO3-)-Met-Gly-D-Trp-Nle-Asp 2-phenylethyl ester 1 (where Nle is norleucine), which is a potent CCK receptor antagonist. In rat and guinea pig dispersed pancreatic acini, this peptide derivative did not alter amylase secretion, but was able to antagonize the stimulation caused by cholecystokinin-related agonists. It caused a parallel rightward shift in the dose-response curve for the stimulation of amylase secretion with half-maximal inhibition of CCK-8-stimulated amylase release at a concentration of about 0.1 microM. Compound 1 was able to inhibit the binding of labeled CCK-9 (the C-terminal nonapeptide of CCK) to rat and guinea pig pancreatic acini (IC50 = 5 X 10(-8) M) as well as to guinea pig cerebral cortical membranes (IC50 = 5 X 10(-7) M). These results indicate that Compound 1 is a potent competitive CCK receptor antagonist.     

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.     

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.     

Differential bile-pancreatic secretory effects of CCK-58 and CCK-8

In this work, we 1) synthesized rat CCK-58, 2) determined the amounts and forms of rat CCK in whole blood after stimulation of its release by casein, 3) determined the potency of CCK-8 and CCK-58 peptides to displace labeled CCK-8 from CCK(A) and CCK(B) receptors transfected into Chinese hamster ovary (CHO) cells, and 4) examined the biological actions of CCK-8 and rat CCK-58 in an anesthetized rat model. CCK-58 was the only detected endocrine form of CCK in rat blood. Synthetic rat CCK-58 was less potent than CCK-8 for displacing the label from CCK(A) and CCK(B) receptors in transfected CHO cells. However, rat CCK-58 was more potent than CCK-8 for stimulation of pancreatic protein secretion in the anesthetized rat. In addition, CCK-58 but not CCK-8 stimulated fluid secretion in this anesthetized rat model. These data suggest that regions outside the COOH terminus of rat CCK-58 influence the expression of CCK biological activity. The presence of only CCK-58 in the circulation and the fact that its biological activity differs from CCK-8 suggests that CCK-58 deserves scrutiny in other physiological models of CCK activity.     

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.     

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.     

Intrinsic photoaffinity labeling probes for cholecystokinin (CCK)-gastrin family receptors. D-Tyr-Gly-[Nle28,31,pNO2-Phe33)CCK-26-33)

Attempts to biochemically characterize the pancreatic cholecystokinin (CCK) receptor by affinity labeling have utilized either 125I-Bolton-Hunter-CCK-33 ("long" probes) or decapeptide analogues of the carboxyl terminus of CCK ("short" probes), and covalent attachment via the amino-terminal regions of these probes. The long probe has identified a protein of Mr = 80,000 while "shorter" probes, which have their site of cross-linking closer to the receptor binding region of the probes, have labeled a distinct protein of Mr = 85,000-95,000. To extend and complement these observations, we have designed and synthesized a new probe for the CCK receptor which incorporates a photolabile p-nitrophenylalanine moiety within the theoretical receptor-binding region of the hormone, as its carboxyl-terminal residue. This "intrinsic" photoaffinity labeling probe has been shown to possess full biological activity, with potency and efficacy in stimulating amylase secretion by dispersed rat pancreatic acini similar to that of CCK-8 (CCK-26-33). When iodinated oxidatively, this probe binds rapidly, in a temperature-dependent, reversible, saturable, specific, high affinity manner to enriched pancreatic plasma membranes. In this work, we have used this probe to specifically label the CCK binding site on rat pancreatic plasma membranes. The Mr = 85,000-95,000 protein previously identified with amino-terminal cross-linking of short probes appears to be the protein labeled with this reagent as well. This provides strong evidence that this pancreatic plasma membrane protein contains the CCK-binding domain of the CCK receptor. This intrinsic photoaffinity labeling probe should be quite useful for the characterization of the active site of this receptor and for other CCK and gastrin receptors in many species.     

Synthesis of biologically active canine CCK-58

The carboxyl terminal octapeptide of cholecystokinin (CCK-8) has been hypothesized to account for the bioactivity of all the molecular forms of cholecystokinin. However, the physiological relevance of CCK-58 has not been rigorously examined because of the lack of sufficient amounts of the peptide and concerns about inactivation of natural peptides during their purification. Therefore, canine-sulfated CCK-58 was synthesized and conditions determined for its unblocking and purification that preserved the sulfated tyrosine. Synthetic CCK-58 was indistinguishable from natural CCK-58 by amino acid analysis and by mass spectrometry. Synthetic CCK-58 and CCK-8 have different patterns of pancreatic stimulation: both caused a dose-related increase in amylase release, while only CCK-58 stimulated bile-pancreatic output volume. Thus, CCK-58 and CCK-8 are biased agonists at the CCK-A receptor (they have distinct patterns of action mediated by the same receptor). Previous work has demonstrated that the identical carboxyl termini of CCK-8 and CCK-58 have different solution conformations. Taken together, the physiological and structural results support the hypothesis that different carboxyl terminal conformations of CCK-58 and CCK-8 alter the expression of their biological activity.     

Characterization of preprocholecystokinin products in the porcine cerebral cortex. Evidence of different processing pathways

Using gel, ion-exchange, and reverse-phase chromatography monitored by radioimmunoassays specific for five sequences of preprocholecystokinin (prepro-CCK), its processing products were measured in neutral and acid extracts of porcine cerebral cortex before and after incubation with trypsin, carboxypeptidase B, and arylsulfatase. Three categories of peptides were found: biologically active peptides, i.e. peptides with the alpha-amidated COOH terminus Trp-Met-Asp-Phe-NH2, comprising large CCKs, i.e. peptides larger than CCK-58 and peptides eluting like CCK-58, CCK-33, and CCK-22; CCK-octapeptides in sulfated and traces of nonsulfated forms; and small CCKs, i.e. traces of CCK-7, large amounts of CCK-5, and modest concentrations of CCK-4 (the structures of CCK-5 and -4 were confirmed by sequence analysis); four NH2-terminal fragments, of which the two predominant ones correspond to the desnonapeptide fragments of CCK-58 and CCK-33; and COOH-terminal extended peptides corresponding to glycine-extended CCK-58, CCK-33, and CCK-8 in small but significant amounts. Thus, in addition to CCK-8 the porcine cerebral cortex synthesizes larger and smaller active CCK peptides in quantities of an order similar to those of CCK-8. The occurrence of these together with the NH2-terminal fragments and glycine-extended peptides can be explained only by the existence of different processing pathways for preproCCK. Consequently, the results suggest that cerebral CCK neurons are heterogeneous and comprise at least three populations with different biosynthetic machineries.     

Estrogens influence cholecystokinin stimulated pancreatic amylase release and acinar cell membrane cholecystokinin receptors in rat.

This study examines the influence of ovariectomy and administration of a pharmacologic dose of estradiol on amylase release from isolated-dispersed rat pancreatic acini and cholecystokinin receptors on rat acinar cell membranes. Rats were sham ovariectomized (intact) or ovariectomized (Ovx) and 21 day timed release pellets containing either estradiol (2.5 mg) or vehicle, were implanted subcutaneously. Eighteen days later, pancreatic acini were isolated from rats by collagenase digestion and differential centrifugation. Total cellular amylase, basal and cholecystokinin octapeptide (CCK8) stimulated amylase release and CCK membrane receptors were measured. Acini isolated from estradiol treated Ovx rats had significantly greater total cellular amylase, compared to acini isolated from either intact or Ovx rats. The amplitude of both total stimulated amylase release and percent total stimulated amylase release were significantly greater for acini isolated from vehicle treated Ovx rats, than acini isolated from either intact or estradiol treated Ovx rats. The magnitude of percent total amylase release of acini isolated from estradiol treated Ovx rats was significantly lower than that of acini isolated from intact rats. Cholecystokinin receptor concentration was significantly greater on membranes prepared from vehicle treated Ovx rats, compared to membranes prepared from either intact or estradiol treated Ovx rats. These data indicate that ovariectomy is associated with increased responsiveness of pancreatic acini to CCK stimulation, while chronic estradiol treatment of ovariectomized rats is associated with increased total cellular amylase and decreased acinar cell responsiveness to CCK8. Estrogen mediated alterations in acinar cell amylase content and amylase release may play a role in estrogen related pancreatitis.     

Preparation of biologically active radioiodinated cholecystokinin for radioreceptor assay and radioimmunoassay.

A modified method is described for the preparation of stable, high specific activity radioiodinated cholecystokinin (CCK) by its conjugation to 125I-Bolton Hunter reagent (125I-BH). Purified radioiodinated CCK (125I-BH-CCK) stimulated amylase release from isolate rat pancreatic acini with a potency identical to that of native CCK. Further, 125I-BH-CCK was highly immunoreactive and reacted with antisera directed toward both the NH2- and COOH-terminal portions of the CCK molecule. Finally, 125I-BH-CCK bound to specific receptor sites on isolated pancreatic acini.     

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.     

Pertussis toxin stimulates cholecystokinin-induced cyclic AMP formation but is without effect on secretagogue-induced calcium mobilization in exocrine pancreas

The role of a pertussis toxin sensitive GTP-binding protein in mediating between cholecystokinin receptors and phosphatidylinositol 4,5-bisphosphate phosphodiesterase as well as in preventing cholecystokinin from increasing cellular cyclic AMP has been investigated using dispersed acini from rabbit pancreas. Pertussis toxin pretreatment (500 ng/ml, 2 h) did not affect cholecystokinin(octapeptide) (CCK-8)-induced increases in cytosolic free Ca2+ as judged from changes in fluorescence obtained from quin2-loaded acini. Although pretreatment with pertussis toxin was also without effect on resting acinar cell cyclic AMP levels, adenylate cyclase activity was increased, since inhibition of cyclic AMP phosphodiesterase activity by isobutylmethylxanthine (IBMX) resulted in an additional increase in cyclic AMP levels in toxin-treated acini, indicating that acinar cell adenylate cyclase activity is under some tonic inhibitory control by the pertussis toxin-sensitive inhibitory GTP-binding protein (Gi) of the adenylate cyclase system. CCK-8 gave an increase in cyclic AMP levels in both control (1.6-fold) and toxin-treated (2.3-fold) acini, leading to cyclic AMP levels in the toxin-treated acini 2-times as high as those in control acini. In the presence of IBMX, the cyclic AMP response to CCK-8 was again markedly enhanced in acini pretreated with the toxin (3.2- vs. 1.8-fold), resulting in cAMP levels in the toxin-treated acini 3.7-times those in the absence of IBMX, 2.5-times those in control acini in the presence of IBMX and 7.0-times those in control acini in the absence of IBMX. Neither the pretreatment with pertussis toxin, nor the presence of IBMX alone, nor the combination had an effect on basal amylase secretion. However, all three treatments potentiated the stimulatory effect of CCK-8 on amylase secretion and the amount of potentiation was proportional to the cyclic AMP levels reached. Our findings suggest that in the intact pancreatic acinar cell Gi inhibition of the catalytic subunit of the adenylate cyclase may largely be responsible for preventing cholecystokinin from increasing cellular cyclic AMP. They moreover show that cyclic AMP is a modulatory agent in rabbit pancreatic enzyme secretion, not able to stimulate secretion itself, but potentiating effects mediated by the phosphatidylinositol-calcium pathway.     

Molecular forms of cholecystokinin in plasma from normal and gastrectomized human subjects following a fat meal

The present study was undertaken to characterize molecular forms of cholecystokinin (CCK) in human fat-stimulated plasma by Sephadex G50 column chromatography followed by radioimmunoassays employing 3 different region-specific antibodies. CCK was extracted and concentrated from plasma of healthy subjects by adsorption to SEP-PAK C18 cartridges and from plasma of gastrectomized patients by addition of 96% ethanol. Antibody 1703 binds to carboxy-terminal CCK-peptides containing at least 14 amino acid residues, antibody T204 to sulfated carboxy-terminal CCK-peptides and antibody 5135 to carboxy-terminal forms of CCK and gastrin. Four molecular forms of CCK were consistently demonstrated; peak I eluted in the void volume and comprised 1.8-10.2% of CCK-immunoreactivity, peak II eluted between the void volume and the CCK-33/39 standard and comprised 9.8-21.6%, peak III eluted at the position of the CCK-33/39 standard and comprised 42.4-55.4%, and peak IV eluted between the CCK-33/39 and CCK-14 standards and comprised 25.4-40.1% of CCK immunoreactivity. Since these 4 molecular forms reacted to all 3 CCK-antibodies it is likely that they contain the sulfated tyrosyl and carboxy-terminal regions of CCK and, therefore, possess biological activity.