NMR evidence for different conformations of the bioactive region of rat CCK-8 and CCK-58

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.     

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.     

Pharmacological examination of cholecystokinin (CCK-8)-induced contractile activity in the rat isolated pylorus

The actions of cholecystokinin (CCK) in the production of a satiety-like state have been suggested to be mediated via receptors for CCK which are located in the pylorus. We investigated the actions of CCK and other pharmacological agents upon the isolated rat pylorus in vitro. We used the change in isometric tension of the tissue preparation (contraction amplitude) as the measure of the effects of the pharmacological agents. Cholecystokinin COOH-terminal octapeptide (CCK-8) was observed to elicit contraction in a dose-dependent manner, with the half-maximal dose (ED50) in the vicinity of 1 nM. Rapid desensitization to CCK was observed. The contraction amplitude was atropine-independent, and was not significantly antagonized by a wide variety of other pharmacological agents. The Na+-channel blocker tetrodotoxin was without effect upon contractile amplitude, as was the K+-channel blocker 4-aminopyridine, except at very high concentrations. Neurotensin, bombesin, and the substance P and bombesin antagonist spantide all elicited contraction in the isolated tissue; neurotensin had a similar potency to CCK-8 and bombesin was 10-15-fold less potent than CCK-8. Unsulfated CCK-8 was at least 170-fold less potent than sulfated CCK-8 and tetragastrin was at least 500-fold less potent than CCK-8. These results suggest that pyloric CCK receptors, which appear to have a pharmacological profile typical of peripheral CCK receptors, may have a physiological role in the peptidergic control of gastric emptying in the rat.     

Identification of nonsulfated cholecystokinin-58 in canine intestinal extracts and its biological properties

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.     

Supramaximal CCK-58 does not induce pancreatitis in the rat: role of pancreatic water secretion

In contrast to supramaximal CCK-8 or caerulein, acute or prolonged supraphysiological levels of endogenous CCK-58 do not cause pancreatitis. Compared with CCK-8, CCK-58 is a much stronger stimulant of pancreatic chloride and water secretion, equivalent to maximally effective secretin, but with a chloride-to-bicarbonate ratio characteristic of acinar fluid. Because supraphysiological endogenous CCK does not cause pancreatitis and because coadministration of secretin ameliorated caerulein- or CCK-8-induced pancreatitis, coincident with restoring pancreatic water secretion, we hypothesized that supramaximal CCK-58 would not induce pancreatitis. Conscious rats were infused intravenously with 2 or 4 nmol x kg(-1) x h(-1) of CCK-8 or synthetic rat CCK-58 for 6 h, and pancreases were examined for morphological and biochemical indexes of acute pancreatitis. A second group was treated as above while monitoring pancreatic protein and water secretion. CCK-8 at 2 nmol x kg(-1) x h(-1) caused severe edematous pancreatitis as evidenced by morphological and biochemical criteria. CCK-58 at this dose had minimal or no effect on these indexes. CCK-58 at 4 nmol x kg(-1) x h(-1) increased some indexes of pancreatic damage but less than either the 2 or 4 nmol x kg(-1) x h(-1) dose of CCK-8. Pancreatic water and protein secretion were nearly or completely abolished within 3 h of onset of CCK-8 infusion, whereas water and protein secretion were maintained near basal levels in CCK-58-treated rats. We hypothesize that supramaximal CCK-58 does not induce pancreatitis because it maintains pancreatic acinar chloride and water secretion, which are essential for exocytosis of activated zymogens. We conclude that CCK-58 may be a valuable tool for investigating events that trigger pancreatitis.     

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.     

Different signaling pathways are involved in CCK(B) receptor-mediated MAP kinase activation in COS-7 cells

Recently, the involvement of the MAP kinase ERK in mitogenic signaling of cholecystokininB (CCK(B)) receptors has been shown. However, the intracellular effector systems involved in this signaling pathway are poorly defined. In this study, we used COS-7 cells transiently transfected with the human CCK(B) receptor to investigate cholecystokinin-induced MAP kinase activation. CCK-8 induced activation of ERK2 which is associated with its phosphorylation and localization in the nucleus. The CCK-8-dependent ERK stimulation is sensitive to wortmannin an inhibitor of phosphoinositide 3-kinases (PI3Ks) indicating the involvement of PI3K activity. To identify the PI3K species involved in mitogenic signaling of the CCK(B) receptor several dominant-negative mutants of PI3K regulatory and catalytic subunits were transiently expressed. Surprisingly, different catalytically inactive mutants of the G protein-sensitive PI3Kgamma did not affect ERK stimulation induced by CCK, whereas a dominant-negative mutant of the regulatory p85 subunit induced significant inhibition of CCK-dependent ERK activity. These results indicate an involvement of PI3K class 1A species alpha, beta or/and delta in signal transduction via CCK(B) receptors. In addition, protein kinase C (PKC)-dependent signaling pathways contribute to CCK(B)-mediated MAP kinase signaling as shown by inhibition of CCK-8-induced ERK activation by the PKC inhibitor bisindolylmaleimide.     

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.     

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.     

Low-affinity CCK-1 receptors inhibit bombesin-stimulated secretion in rat pancreatic acini--implication of the actin cytoskeleton

EXPERIMENTAL OBJECTIVES: Stimulation of low-affinity CCK-1 receptors on pancreatic acini leads to inhibition of enzyme secretion. We studied signal transduction mechanisms to identify potential causes for the reduced secretion. RESULTS: Co-stimulation experiments with CCK, CCK-JMV-180, and bombesin revealed an inhibition of bombesin-stimulated enzyme secretion by low-affinity CCK-1 receptors. Binding of 125I-gastrin-releasing peptide (the mammalian analogue of bombesin) to acini after CCK preincubation was not altered. After a short preincubation of acini with high concentrations of CCK, intracellular calcium remained responsive to bombesin. In contrast to bombesin or CCK at concentrations of 10(-10) M or lower, high concentrations of CCK caused a strong activation of p125 focal adhesion kinase (p125(FAK)) and a marked reorganisation of the actin cytoskeleton. CONCLUSIONS: Inhibitory mechanisms triggered by low-affinity CCK-1 receptors interrupt enzyme secretion from pancreatic acini at late stages in the signal transduction cascades since bombesin receptor binding and early signalling events remained intact after CCK preincubation. A reorganisation of the actin cytoskeleton is suggested to be the mechanism by which low-affinity CCK-1 receptors actively interrupt enzyme secretion stimulated by other receptors.     

CCK2 (CCK(B)/gastrin) receptor mediates rapid protein kinase D (PKD) activation through a protein kinase C-dependent pathway

Addition of gastrin or cholecystokinin octapeptide (CCK-8) to cultures of Rat-1 cells stably transfected with the CCK2 (CCK(B)/gastrin) receptor induced protein kinase D (PKD) activation that was detectable within 1 min and reached a maximum ( approximately 10-fold) after 2.5 min of hormonal stimulation. Half-maximal PKD activation for both CCK-8 and gastrin was achieved at 10 nM. Treatment with various concentrations of the selective PKC inhibitors Ro 31-8220 or GF-I potently blocked PKD activation induced by subsequent addition of CCK-8 in a concentration-dependent fashion. Our results indicate that PKC-dependent PKD activation is a novel early event in the action of gastrin and CCK-8 via CCK2 receptors.     

Role of cholecystokinin in the intestinal phase of pancreatic circulation in dogs

The regulatory mechanisms of postprandial pancreatic hyperemia are not well characterized. The aim of this study is to clarify the role of cholecystokinin (CCK) in the intestinal phase of pancreatic circulation. Pancreatic, gastric, and intestinal blood flows were measured by ultrasound transit-time blood flowmeters in five conscious dogs. Pancreatic and gastric secretion and blood pressure were also monitored. Synthetic CCK octapeptide (CCK-8) or gastrin heptadecapeptide (gastrin-17) was infused intravenously, and milk was infused into the duodenum with or without loxiglumide, a specific CCK-A receptor antagonist. CCK-8 induced dose-related increases of pancreatic, but not gastric or intestinal, blood flow and protein secretion without affecting systemic blood pressure. Gastrin-17 did not affect pancreatic blood flow. An intraduodenal infusion of milk increased pancreatic and intestinal blood flows and pancreatic protein secretion. Loxiglumide completely inhibited pancreatic blood flow and protein responses to CCK-8 and milk but not the intestinal blood flow response. CCK is a potent and specific pancreatic vasodilator, with its effect mediated by CCK-A receptors. CCK plays an important role in the regulation of the intestinal phase of the pancreatic circulation in dogs.     

Isolation and characterization of sulphated and nonsulphated forms of cholecystokinin-58 and their action on gallbladder contraction.

Cholecystokinin (CCK) exists in multiple molecular forms with different polypeptide lengths and the absence or presence of sulphation. We have isolated sulphated and nonsulphated forms of CCK-58 from porcine intestine and have determined their bioactivities in a guinea-pig gallbladder contraction assay. Both forms co-eluted in cation-exchange chromatography and in several rounds of reverse-phase (RP)-HPLC, but separated upon RP-HPLC using a water/acetonitrile system with heptafluorobutyric acid as counter ion. Nonsulphated CCK-58 was the form detected by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry because of desulphation in that process. The biological activity of CCK-58 and CCK-33 is equipotent, although the kinetics of the response differ. Sulphated CCK-58 was found to be 35 times more potent than nonsulphated CCK-58. In contrast, sulphated CCK-8 is 150 times more potent than nonsulphated CCK-8, and for sulphated and nonsulphated CCK-33, the activities differ by a factor of 100. This type of correlation indicates that the N-terminal end of CCK-58 partially compensates for the decrease in activity arising from the lack of sulphated tyrosine. Given its fairly high bioactivity, nonsulphated CCK-58 may have a physiological significance.     

Crucial role of position 40 for interactions of CCK-58 revealed by sequence of cat CCK-58

Evidence suggests that amino terminal extensions of CCK-8 affect the carboxyl terminal bioactive region of CCK. Cat CCK-58 was purified by low pressure reverse phase and ion-exchange chromatography steps and several reverse phase HPLC steps. The purified peptide and its tryptic fragments were characterized by mass spectral analysis and microsequence analysis. The structure of cat CCK-58 is: AVQKVDGEPRAHLGALLARYIQQARKAPSGRMSVIKNLQSLDPSHRISDRDY(SO3) MGWMDF-amide. Cat and dog CCK-58 are identical except for position 40 which is serine in cat and asparagine in dog. Radioimmunoassay detected cat CCK-58 about 1/10th as well as dog CCK-58, indicating a marked effect on C-terminal immunoreactivity. Cat CCK-58 with a serine at position 40, the same residue found in pig, mouse, cow and rabbit CCK-58, can be used as a unique bioprobe for defining how amino terminal amino acids influence the structure and bioactivity of the carboxyl terminal region of CCK.     

CCK-58: A novel reagent for studying the regulation of cholecystokinin bioactivity

CCK-58 has been shown to be the major circulating form of the hormone in the dog and human. To date, there have been no reports on its biological activity in vivo. We report here that CCK-8 and CCK-58 were equipotent in decreasing gastric motor function after bolus doses and in stimulating protein secretion after continuous infusion in urethane-anesthetized rats. The present results are the first on the in vivo activity of CCK-58, and indicate that because CCK-58 is equipotent to CCK-8, and because it is a major released and circulating form, it may be considered as a major contributor to the expression of cholecystokinin bioactivity.     

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.     

Functional and molecular characterization of CCK receptors in the rat pancreatic acinar cell line AR 4-2J.

Competitive inhibition binding studies on membranes from the rat pancreatic AR 4-2J cell line revealed the predominance (80%) of low selectivity CCK receptors (KD of 1 nM and 4 nM for, respectively, CCK-8 and gastrin-17I (G-17I] over selective receptors (20% with a KD of 1 nM and 1 microM for, respectively, CCK-8 and G-17I). Amylase secretion was stimulated by low concentrations of CCK-8, G-17I and CCK-4. G-17I-induced amylase secretion was unaffected by 100 nM of the selective peripheral CCK-A receptor antagonist L-364,718, suggesting that amylase hypersecretion followed non-selective CCK receptor activation, a function normally assumed by selective CCK-A receptors in rat pancreatic acini. Direct ultraviolet irradiation of AR 4-2J cell membranes preloaded with 125I-BH-CCK-33 or 125I(Leu)G(2-17)I resulted in covalent cross-linking with, respectively, a 90 kDa protein and a 106 kDa protein, both distinct from the 81 kDa CCK binding species revealed in normal rat pancreatic membranes. Gpp[NH]p increased the dissociation rate of CCK-8 and G-17I from AR 4-2J cell membranes, indicating a coupling of receptors with guanyl nucleotide regulatory protein(s) G. [32P]ADP-ribosylation of AR 4-2J cell membranes allowed to detect the presence of two Gs alpha (the 50 kDa form predominating over the 45 kDa form) and one Gi alpha (41 kDa). However, Gi and Gs may not be involved in gastrin stimulation of amylase secretion, as Bordetella pertussis toxin and cholera toxin pretreatment of cells did not suppress G-17I-dependent amylase secretion.     

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.     

Unsulfated CCK-8 not blocked by proglumide or CR 1409 in the mouse abdominal irritant-induced stretching assay: possible central site of action

Previous studies have shown that unsulfated cholecystokinin octapeptide (CCK-8-U) shares with the sulfated octapeptide (CCK-8-S) and the carboxyl-terminal tetrapeptide (CCK-4) the ability to block abdominal irritant-induced stretching when administered intracerebroventricularly. The effects of CCK-8-U, however, are not naloxone-reversible and do not appear upon systemic administration. To assess the hypothesis that the antistretching effects of CCK-8-U are mediated by central-type (CCK-B), rather than peripheral-type (CCK-A) receptors, the present experiments examined the reversal of these effects by CR 1409, a CCK receptor antagonist with in vitro selectivity for CCK-A receptors, and by proglumide. Both antagonists, when administered ICV, blocked the antistretching effects of CCK-8-S and CCK-4 (ICV), but not those of CCK-8-U. CR 1409 was approximately 40 times more potent against CCK-8-S by the ICV route than subcutaneously, indicating a likely action on CCK-A receptors in the brain. The effects of morphine, bombesin and neurotensin (ICV) were not blocked by CR 1409, indicating specificity for CCK receptor-mediated effects. The antistretching effects of CCK-8-U do not appear to be mediated by CCK-A receptors, and the possibility of a CCK-B receptor site of action must be considered.     

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.