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A novel molecularly imprinted polymer (MIP) monolith for highly selective extraction of cholecystokinin (CCK) neuropeptides was prepared in a micropipette tip. The MIPs were synthesized by epitope imprinting technique and the polymerization conditions were investigated and optimized. The synthesized MIPs were characterized by infrared spectroscopy, elemental analyzer and scanning electron microscope. A molecularly imprinted solid-phase microextraction (MI-μ-SPE) method was developed for the extraction of CCK neuropeptides in aqueous solutions. The parameters affecting MI-μ-SPE were optimized. The results indicated that this MIP monolith exhibited specific recognition capability and high enrichment efficiency for CCK neuropeptides. In addition, it showed excellent reusability. This MIP monolith was used for desalting and enrichment of CCK4, CCK5 and CCK8 from human cerebrospinal fluid prior to matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis, and the results show that this MIP monolith can be a useful tool for effective purification and highly selective enrichment of multiple homologous CCK neuropeptides in cerebrospinal fluid simultaneously. By employing MI-μ-SPE combined with HPLC-ESI-MS/MS analysis, endogenous CCK4 in human cerebrospinal fluid was quantified., (Copyright © 2015 John Wiley & Sons, Ltd.)

As a group of intestinal hormones and neurotransmitters, cholecystokinins (CCKs) regulate and affect pancreatic enzyme secretion, gastrointestinal motility, pain hypersensitivity, digestion and satiety, and generally contain a DYMGWMDFG sequence at the C-terminus. Many CCKs have been reported in mammals. However, only a few have been reported in amphibians, such as Hyla nigrovittata, Xenopus laevis, and Rana catesbeiana, with none reported in urodele amphibians like newts and salamanders. Here, a CCK called CCK-TV was identified and characterized from the skin of the salamander Tylototriton verrucosus. This CCK contained an amino acid sequence of DYMGWMDF-NH2 as seen in other CCKs. A cDNA encoding the CCK precursor containing 129 amino acid residues was cloned from the cDNA library of T. verrucosus skin. The CCK-TV had the potential to induce the contraction of smooth muscle strips isolated from porcine gallbladder, eliciting contraction at a concentration of 5.0 x 10⁻¹¹ mol/L and inducing maximal contraction at a concentration of 2.0 x 10⁻⁶ mol/L. The EC50 was 13.6 nmol/L. To the best of our knowledge, this is the first report to identify the presence of a CCK in an urodele amphibian.

Most proteins undergo posttranslational modifications that govern the function of the protein. In synchrony, correspondingly unmodified proteins that are functionally silent or act differently may also be synthesized. The gut hormone precursor, procholecystokinin (proCCK) is an example of a protein that is heavily modified. An essential modification is O-sulfation of Y₇₇, which is necessary for the gallbladder emptying effect of CCK peptides via the CCKA-receptor. In order to examine possible in vivo synthesis also of nonsulfated CCK, we have established a two-step analysis that requires tryptic cleavage at a defined processing site in proCCK (R₇₅-D₇₆) followed by monospecific RIA-measurement of the then exposed nonsulfated N-terminal sequence of CCK-8 (DYMGW…). The analysis shows that endocrine cells in the gut synthesize nonsulfated CCK peptides (-58, -33, -22, and -8) in the order of 20-35% of the corresponding sulfated CCKs. Since nonsulfated CCK peptides are full agonists of the CCKB-receptor, the assay has revealed a hitherto unrecognized gut hormonal peptide system. The assay may prove useful in the diagnosis and control of diseases with hyperCCKemia. This includes CCK-producing neuroendocrine tumors such as the recently described CCKomas and medullary thyroid C-cell carcinomas.

Background: Numerous studies have demonstrated that in vitro controlled enzymatic hydrolysis of fish and shellfish proteins leads to bioactive peptides. Ultrafiltration (UF) and/or nanofiltration (NF) can be used to refine hydrolysates and also to fractionate them in order to obtain a peptide population enriched in selected sizes. This study was designed to highlight the impact of controlled UF and NF on the stability of biological activities of an industrial fish protein hydrolysate (FPH) and to understand whether fractionation could improve its content in bioactive peptides., Results: The starting fish protein hydrolysate exhibited a balanced amino acid composition, a reproducible molecular weight (MW) profile, and a low sodium chloride content, allowing the study of its biological activity. Successive fractionation on UF and NF membranes allowed concentration of peptides of selected sizes, without, however, carrying out sharp separations, some MW classes being found in several fractions. Peptides containing Pro, Hyp, Asp and Glu were concentrated in the UF and NF retentates compared to the unfractionated hydrolysate and UF permeate, respectively. Gastrin/cholecystokinin-like peptides were present in the starting FPH, UF and NF fractions, but fractionation did not increase their concentration. In contrast, quantification of calcitonin gene-related peptide (CGRP)-like peptides demonstrated an increase in CGRP-like activities in the UF permeate, relative to the starting FPH. The starting hydrolysate also showed a potent antioxidant and radical scavenging activity, and a moderate angiotensin-converting enzyme (ACE)-1 inhibitory activity, which were not increased by UF and NF fractionation., Conclusion: Fractionation of an FPH using membrane separation, with a molecular weight cut-off adapted to the peptide composition, may provide an effective means to concentrate CGRP-like peptides and peptides enriched in selected amino acids. The peptide size distribution observed after UF and NF fractionation demonstrates that it is misleading to characterize the fractions obtained by membrane filtration according to the MW cut-off of the membrane only, as is currently done in the literature., (Copyright (c) 2010 Society of Chemical Industry.)

Recombinant human progastrin(6-80) binds two ferric ions with an apparent dissociation constant of 2.2 +/- 0.1 microM [Baldwin (2004) Protein J 23:65-70]. The aims of the present study were to express fragments of recombinant procholecystokinin and to determine whether or not they bound ferric ions. Recombinant rat and human procholecystokinin(57-95) were expressed as glutathione S-transferase fusion proteins in E. coli. The fusion proteins were bound to glutathione-agarose, cleaved with thrombin, and purified by reverse phase HPLC. Recombinant procholecystokinin(57-95) did not bind to either the CCK1 or CCK2 receptor with high affinity. No change in absorption spectrum was observed on addition of ferric ions, and analysis of the quenching of tryptophan fluorescence observed in the presence of ferric ions indicated that binding to procholecystokinin(57-95) was at least 40-fold weaker than the binding of ferric ions to progastrin(6-80).

Many neuroendocrine peptides that are distributed in amphibian gastrointestinal tract and central nervous system are also found in amphibian skins, and these peptides are classified into skin-gut-brain triangle peptides, such as bombesins, gastrin-releasing peptides. Cholecystokinins (CCKs) are neuroendocrine peptides known for their production in the gastrointestinal tract and central nervous system of mammalians. Several CCKs have been identified from two amphibians, Rana catesbeiana and Xenopus laevis. These amphibian CCKs are found to be express in brain and in the gastrointestinal tract, but not in skin. In the current report, a cholecystokinin (CCK) isoform was identified from skin secretions of the frog, Rana nigrovittata. Its amino acid sequence is RVDGNSDQKAVIGAMLAKDLQTRKAGSSTGRYAVLPNR PVIDPTHRINDRDYMGWMDF, which is the same with that of CCK from R. catesbeiana. Four different cDNAs (GenBank accession nos. EF608063-6) encoding CCK precursors were cloned from the cDNA library of the skin of R. nigrovittata. The present data demonstrated that amphibian CCK could also be expressed in gastrointestinal tract, central nervous system and skin as other amphibian skin-gut-brain triangle peptides.

Objectives: We propose that regions outside the bioactive 7-amino acid carboxyl terminus of cholecystokinin (CCK)-58 influence its biological activity. Here we evaluate if sequence variation of the N-terminal regions of rabbit and canine CCK-58 changes their biological activities., Methods: Cholecystokinin-like immunoreactivity was purified from rabbit intestinal extracts by reverse phase and ion-exchange high-performance liquid chromatography steps. The peptide was characterized by microsequence and mass spectral characterizations of the intact and tryptic peptides. Canine and rabbit CCK-58 were evaluated for their CCK1 and CCK2 receptor binding, receptor activation, and immunologic properties., Results: The sequence of rabbit CCK-58 differs from that of canine CCK-58 in 9 of the amino terminal 40 residues. Canine CCK-58 was approximately 3-fold more potent than rabbit CCK-58 for CCK1 receptor binding and CCK2 receptor binding, but about the same potency for stimulation of amylase release from purified acinar cells. The canine peptide was 9-fold more immunoreactive than rabbit CCK-58., Conclusions: Canine and rabbit CCK-58 have different biological and immunologic properties that can only result from differences in their N-terminal sequences which influence the properties of their identical carboxyl termini. These results are the first direct demonstration that amino acids outside the C-terminus of CCK-58 influence CCK biological activity.

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.

The precursor of cholecystokinin (pro-CCK) was expressed and purified from media of stably transfected D.Mel-2 cell as an V5-His tagged fusion protein. Its identity was confirmed using SDS-PAGE, immunoblotting, gel filtration chromatography, HPLC, and Mass Spectroscopy. Two major forms of pro-CCK were found with a molecular weight of about 14.4 and 11.3 kDa. The smaller form represents the V5-His tagged pro-CCK after cleavage at a single arginine residue at CCK-58. This cleavage is probably being performed by endogenous proteases in these cells. Purification of the desired larger form of pro-CCK is possible using a nickel column with a recovery of about 20%, yielding 500 microg/L media. The purified protein is stable for several months and can be used for further functional studies of pro-CCK.

To identify the teleost gastrin, CCK/gastrin family genes were isolated from puffer and flounder. The cDNA of puffer gastrin, CCK1 and CCK2 were 678, 752 and 533bp, respectively. Puffer gastrin gene consists of three exons, in contrast, CCKs consist of four exons. Flounder gastrin mRNA (526bp) was expressed in the intestine but not in the brain. It was developed synchronously with the stomach differentiation in the larval stage. The phylogenetic analysis shows that puffer and flounder gastrin classified into the vertebrate gastrin cluster and two types of CCK were probably produced by the genome duplication occurred in teleost phylogeny.

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.

The distribution of cholecystokinin and gastrin-like immunoreactive cell bodies and fibers in the nervous system of 2 annelid worms, Lumbricus terrestris and Eisenia fetida, has been studied by means of immunohistochemistry. The cerebral ganglion contains 170-250, the subesophageal ganglion contains 120-150, and the ventral ganglia contain 50-75 cholecystokinin immunoreactive cells, that represent 8-12%, 8-10% and 4-5% of the total cell number, respectively. The anti-gastrin serum stained 330-360 nerve cells in the cerebral, 32-46 in the subesophageal and 7-25 in the ventral cord ganglia, representing 15-16%, 2-3% and 0.5-2% of the total cell number. Immunopositivity was found with both antisera in the enteric nervous system, where the stomatogastric ganglia and the enteric plexus contain immunoreactive cells and fibers. Immunopositive cells were found in the epithelial and subepithelial cells, as well as in nerve cells innervating the muscular layer of the gastrointestinal tube. Various epidermal sensory cells also displayed strong immunoreactivity. According to our findings and the results of several functional studies, it is suggested that in annelids cholecystokinin- and gastrin-like peptides may be involved in digestive regulation, sensory processes and central integrating processes.

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.

Cholecystokinins from brain and small intestine of the bullfrog (Rana catesbeiana) and red-eared slider turtle (Pseudomys scripta) were isolated. The purifications were monitored by an antiserum specific for the common C-terminus of mammalian cholecystokinin and gastrin. The peptide structures were identified by sequence analysis of the intact peptides and proteolytic fragments, mass spectrometry, and amino acid analysis. Brain and small intestine of both species contained cholecystokinin-8 and substantial amounts of cholecystokinin-7. Furthermore, the small intestine of both frog and turtle contained a major fraction of the immunoreactive material as large peptides consisting of 69 residues and 70 residues, respectively. The structure for frog cholecystokinin-69 is ASSSAQLKPFQRIDGTSDQKAVIGAMLAKYLQTRKAGSSTGRYAVLPNRPVIDPTHRINDRDYMGWMDF .NH2 and the structure for turtle cholecystokinin-70 is VPSSAGQLKPIQRLDGNVDQKANIGALLAKYLQQARKGPTGRISMMGNRVQNIDPTHRINDRDYMGWMD F.NH2. All the isolated peptides were tyrosine sulfated at the seventh last residue. The peptides are highly similar to each other and to mammalian cholecystokinins (70% mutual identity and more than 50% identity with human cholecystokinin). Thus, they are clearly related to the known mammalian cholecystokinins. Both peptides include the monobasic and dibasic cleavage sites giving rise to cholecystokinins-33, -39, and -58 in mammals. However, only a small amount of turtle cholecystokinin-40 (corresponding to mammalian cholecystokinin-39) was isolated. This confirms that post-translational processing is highly species dependent. Recently, we isolated peptides from frog and turtle antrum. Following their origin they were named gastrins in spite of their C-terminal cholecystokinin-like structure. Thus, two different cholecystokinin/gastrin peptides exist in frog and turtle justifying the choice of two names. This finding of two members of the cholecystokinin/gastrin family in frog shows that the divergence of cholecystokinin and gastrin occurred simultaneously with or earlier than the appearance of amphibia during phylogenesis. Frog cholecystokinin and gastrin show sufficient similarity along the whole sequence to support the notion of a gene duplication of a common ancestor.

Reversed-phase chromatography was used to separate several forms of cholecystokinin-like peptides (CCKLP) from the pericardial organs (PCOs) of the spiny lobster Panulirus interruptus. Fast protein liquid chromatography of PCOs, stomatogastric ganglia (STGs) and eyestalks revealed five peaks of CCKLP (peaks A-E) that were common to all three tissues, as well as two additional peaks (peaks F and G) in the STG. Peaks A-E were present in the hemolymph of fed, but not starved, lobsters. The bioactivity of peaks A-E was tested on the gastric mill rhythm of the isolated STG. Only peak E elicited activity. The effects of peak E included activating the gastric mill rhythm in quiescent preparations and strengthening existing rhythms in a dose-dependent manner. Further purification of peak E by high performance liquid chromatography resolved this peak into two immunoreactive peaks, one of which retained its bioactivity. The effects of peak E were blocked by the CCK antagonist proglumide. These results are consistent with a role for peak E in the feeding-induced activation of the gastric mill.

The cholecystokinin (CCK)- and tyrosine hydroxylase (TH)-like immunoreactive (LI) axons and boutons were studied in the caudal and medial parts of the rat nucleus accumbens (NAC), using the indirect immunoperoxidase technique, at the electron microscopic level. Both CCK- and TH-LI boutons contained clear synaptic vesicles and large granular vesicles of similar size, but the CCK-LI boutons contained more large granular vesicles than TH-LI boutons. The CCK-LI and TH-LI boutons were heterogeneous. This finding might be related to the various immunoreactive neuronal types innervating the caudomedial NAC. However, the CCK-LI boutons (containing mostly small, round, clear synaptic vesicles) formed mainly asymmetrical synaptic contacts with dendritic spines whereas the TH-LI boutons (containing medium-sized as well as small, round, clear synaptic vesicles) formed mostly symmetrical synaptic contacts with dendritic shafts.

The coexistence of serotonin and cholecystokinin was studied in foetal sheep lungs at pseudoglandular stage of development by light microscopic immunohistochemistry. The coexistence was examined by staining consecutive sections with the different antibodies. Serotonin and cholecystokinin immunoreactivity was found within consecutive sections of most bronchopulmonary neuroepithelial bodies and in consecutive sections of the same intrapulmonary autonomic ganglia.

Background: Cholecystokinin (CCK) 58 is the predominant molecular form of CCK in canine and human intestine and circulating blood. There is no report on the metabolism and clearance rate of CCK-58. The aim of this study was to compare the in vivo half-life and metabolism of CCK-58 with that of synthetic CCK-8., Methods: CCK-58 was purified from canine intestine by consecutive high-performance liquid chromatographic (HPLC) and fast protein liquid chromatographic steps. The peptides were given to 12 dogs as an intravenous (IV) bolus injection to determine the half-life of circulating CCK. Six dogs were given CCK-58 or CCK-8 as a constant IV infusion to determine plasma clearance rates and stability in circulating blood. Circulating molecular forms of CCK were determined by radioimmunoassay after extraction of CCK from plasma and characterization by HPLC., Results: The half-life of CCK-58 was 4.4 +/- 0.6 minutes compared with 1.3 +/- 0.1 minutes for CCK-8. Less than 5% of CCK-58 could be detected as smaller forms during constant IV infusion., Conclusions: The longer half-life of CCK-58 compared with CCK-8 and the minimal conversion into smaller forms during constant IV infusion are consistent with the finding that CCK-58 is not only the major stored form but also the circulating form of CCK after endogenous stimulation in dogs.

The synthesis of [Phe(p-CH2SO3Na)52, Nle32,53,56 Nal55]-CCK20-58, [Tyr52, Nle32,53,56, Nal55]-CCK-58 and of [Phe(p-CH2SO3Na)52, Nle32,53,56, Nal55]-CCK-58 using the (9-fluorenylmethyloxy)-carbonyl (Fmoc) strategy on a 2,4-DMBHA resin is described. The crude peptide preparations were extremely complex when analyzed by RP-HPLC, capillary zone electrophoresis (CZE), and ion-exchange chromatography (IE-FPLC). We found that the most effective strategy for purification included cation-exchange chromatography followed by a RP-HPLC desalting step. The highly purified peptides (purity greater than 90%) were characterized by RP-HPLC, size exclusion HPLC (SEC), IE-FPLC, CZE, mass spectrometry, amino acid analysis, and Edman sequence analysis (for [Tyr52, Nle32,53,56, Nal55]-CCK-58). The results demonstrate the applicability of the 2,4-DMBHA resin for Fmoc solid-phase synthesis of long peptides amides (58 residues in length in this case) as well as the efficacy of an FPLC/RP-HPLC approach for the purification of very long, heterogeneous crude peptides, allowing a true assessment of the biological properties of these analogs to be carried out. [Phe(p-CH2SO3Na)52, Nle32,53,56, Nal55]-CCK20-58 was less than 1% as potent as CCK-8 while [Tyr52, Nle32,53,56, Nal55]-CCK-58 and [Phe(p-CH2SO3Na)52, Nle32,53,56, Nal55]-CCK-58 were inactive at the doses tested (< 0.01%).

From porcine duodenal mucosa we have identified three major procholecystokinin (proCCK) fragments: desoctaCCK-33, desnonaCCK-33 and desnonaCCK-39. (DesoctaCCK-33 means CCK-33 devoid of the 8 C-terminal amino acids, etc.). The fragments were purified by immunoaffinity chromatography and three steps of reverse phase HPLC monitored by a radioimmunoassay specific for the N-terminal part of CCK-33. The structures could be deduced from the proCCK sequence by N-terminal sequence determination and mass spectrometry. Whereas desnona-fragments of CCK have been described before, this is the first finding of a desoctaCCK, and it indicates that CCK-8 is released from the longer forms by endogenous cleavage of the Arg-Asp-bond. A carboxypeptidase B-like exopeptidase subsequently must produce the desnona-fragments by removing the arginine residue.

The release of cholecystokinin from the dorsal and ventral region of the rat bed nucleus of stria terminalis was studied. Minislices from both regions were superfused with Krebs-Ringer-phosphate, and the cholecystokinin released into the physiological medium was concentrated previous to radioimmunoassay determination. For this purpose, cholecystokinin was adsorbed onto a C18 reverse-phase column and eluted with acetonitrile. Cholecystokinin standards (10-50 pg) were subjected to the above procedure, which allowed a 20- to 50-fold concentration of the peptide with an 80% recovery. Potassium-induced release of cholecystokinin from minislices of dorsal and ventral regions of the bed nucleus of stria terminalis was measured successfully using the above procedure to concentrate the peptide. Lesion of the stria terminalis, a fiber tract originating in the amygdala, provoked a significant decrease in cholecystokinin levels in the ventral region of the bed nucleus of strial terminalis. Thus, cholecystokinin released from minislices of the ventral region of the stria terminalis may be of amygdaloid origin.

In the radioimmunoassay of human plasma cholecystokinin (CCK) using an antiserum OAL-656, it has been difficult to get stable fasting values as well as prominent postprandial increase. Therefore, we tried to extract human plasma with ethanol before the radioimmunoassay, and could solve these problems, that is fasting concentrations became stable (16.7 +/- 2.5 pg/ml) (mean +/- S.D., n = 9), and CCK concentrations were augmented promptly after feeding and were kept at higher levels for a certain period.

The localization and distribution of neuropeptides in the central nervous system of the pig roundworm, Ascaris suum, have been determined by an indirect immunofluorescence technique in conjunction with confocal microscopy. Antisera to 25 vertebrate peptides and two invertebrate peptides were used to screen the worm for immunoreactivity (IR). Immunostaining was obtained with antisera to pancreatic polypeptide (PP), peptide YY (PYY), neuropeptide Y (NPY), gastrin, cholecystokinin (CCK), substance P (SP), atrial natriuretic peptide (ANP), salmon gonadotropin-releasing hormone (SGnRH), mammalian gonadotropin-releasing hormone (MGnRH), chromogranin A (CGA) and FMRFamide. The most extensive patterns of IR occurred with antisera to PYY, FMRFamide and gastrin. IR was evident in nerve cells and fibres in the ganglia associated with the anterior nerve ring and in the main nerve cords and their commissures; IR to FMRFamide also occurred in the posterior nerve ring. Immunostaining for the other peptides was confined to the nerve cords, with the number of immunoreactive nerve fibres varying from peptide to peptide.

In human, cholecystokinin-immunoreactive nerve fibres are abundantly distributed in the ventral striatum and in other regions of the basal ganglia interconnected with the limbic system. This suggests that this neuropeptide is involved in the initiation of movements following emotional stimuli. The demonstration of an extended cholecystokinin expression in cortico-, thalamo-, and nigrostriatal pathways using in situ hybridization indicates that this neuromodulator may have some important functions either in motor, cognitive or limbic systems. In addition, disruption of the nigrostriatal dopaminergic pathway induces the expression of cholecystokinin in the striatal neurons. This new example of functional plasticity suggests that dopamine exerts a negative tonus on cholecystokinin expression. Finally, cholecystokinin expression is increased in meso-limbic and nigrostriatal dopaminergic neurons of schizophrenic patients.

A reproducible, quantitative isoelectric focusing method using capillary electrophoresis that exhibits high resolution and linearity over a wide pH gradient was developed. RNase T1 and RNase ba are two proteins that have isoelectric points (pI's) at the two extremes of a pH 3-10 gradient. Site-directed mutants of the former were separated from the wild-type form and pI's determined in the same experiment. The pI's of RNase T1 wild-type, its three mutants, and RNase ba were determined for the first time as 2.9, 3.1, 3.1, 3.3, and 9.0, respectively. The paper describes the protocol for isoelectric focusing by capillary electrophoresis, as well as presenting data describing the linearity, resolution, limits of mass loading, and reproducibility of the method.

So far, no efficient affinity chromatography for CCK receptor purification has been reported that prevented obtention of sequenceable amounts of purified receptor. In this work, 10% of plasma membrane receptor sites were specifically cross-linked with the photoreactive cleavable agonist 125I-ASD-[Thr28, Ahx31]-CCK-25-33, solubilized by NP-40, chromatographied on immobilized wheat germ agglutinin and further immunopurified using anti-CCK antibodies to an overall rate of 3000-3600-fold. Analysis of eluted material demonstrated a protein migrating at Mr 85,000-100,000 and the absence of 35S-labeled impurity. This single and efficient affinity chromatography should provide enough homogeneous receptor protein for microsequence determination and leads to consider immunoaffinity chromatography on immobilized anti-ligand antibodies as a potential tool for purification of membrane receptors.

An 83-amino acid cholecystokinin peptide with a sulfated tyrosine and an amidated carboxyl terminus (CCK-83) was purified from human intestinal mucosa. The purified peptide was chemically characterized, and its bioactivity was compared to CCK-8. Several post-translational processing steps such as cleavage at basic residues, sulfation, and amidation are necessary to form biologically active cholecystokinin from its nascent prepropeptide. The discovery of CCK-83 gives new insight into the order of preprohormone processing. The processing of prepro-CCK appears to be in the order of: 1) signal peptidase cleavage, 2) tyrosine sulfation, 3) cleavage after a carboxyl-terminal pair of basic residues, 4) carboxypeptidase B-like cleavage of these basic residues, 5) amidation (which results in the formation of CCK-83), and 6) cleavage at monobasic residues by endopeptidases (which results in the smaller molecular forms of cholecystokinin). The characterization of biologically active CCK-83 with a sulfated tyrosine and an amidated carboxyl terminus establishes the site of signal peptidase action and suggests an order of post-translational modifications that give rise to the various molecular forms of cholecystokinin.

Cholecystokinin analogues containing N-methyl amino acids were studied by reversed-phase high-performance liquid chromatography at reduced temperatures. A reduction in temperature to -17 degrees C led to lower efficiency, but at the same time separations of cis and trans isomers (and some impurities) were achieved. The velocity constants for cis-trans equilibria were calculated.

Four gastrin/cholecystokinin-like peptides (G/CCK) which cross-react with a specific C-terminal gastrin/CCK antiserum have been isolated from the stomach of the marine crustacean Nephrops norvegicus. The molecular weight of the four peptides was estimated between 1000 and 2000 Da by molecular sieving. By radioimmunoassay, the cross-reactivity of these peptides with human gastrin 17-I was found to be around 0.03%. Pure peptidic fractions were recovered after four successive steps of HPLC. Amino-acid analysis suggested a similarity between the four peptides identified which may belong to a new family. A limited homology between the C-terminus of one Nephrops peptide and vertebrate G/CCK was found after sequencing. Two of the peptides exhibited secretagogue effects on crustacean isolated midgut glands. The Nephrops peptides, although structurally distinct from the vertebrate G/CCKs, appear to serve similar biological functions in crustaceans.

We have studied the production and release of cholecystokinin (CCK) forms by rat medullary thyroid cancer (MTC) in vivo. MTC cells were inoculated s.c. into 8 Wag-Rij rats. One month later, after i.v. injection of calcium plasma levels of CCK, calcitonin and tissue contents of gastrin and calcitonin were determined by radioimmunoassay (RIA), immunocytochemistry, and high-pressure liquid chromatography (HPLC). The tumors were passed 4 times to 8 rats in 1-month intervals fasting levels of CCK in control rats were unaffected by calcium stimulation, and in tumor-bearing rats, plasma CCK was elevated in 3 out of 4 passages, falling to normal levels at the end of passage 4. Hypercalcemia had no effect on plasma levels of CCK in tumor-bearing rats, but did stimulate the release of calcitonin in both control and some tumor-bearing rats in later passages. CCK-8 sulfate was found in all 4 tumor passages but not CCK-33/39. We conclude that rat MTC synthesizes and releases CCK-8, but unlike calcitonin, release of CCK appears unresponsive to calcium stimulation.

Expression of preprocholecystokinin mRNA was studied in regions of post mortem human brain using RNA blot analysis (Northern blot) and in situ hybridization. Northern blot analysis using a cDNA probe showed high levels of an approximately 0.8 kb preprocholecystokinin mRNA in all regions of neocortex examined. Lower levels of preprocholecystokinin mRNA were detected in amygdaloid body and thalamus. In situ hybridization analysis using the same cDNA probe revealed numerous weakly labelled neurons in different areas of human neocortex and less numerous neurons in hippocampus and amygdaloid body. High-performance liquid-chromatography and gel-chromatography combined with radioimmunoassay of cholecystokinin-like immunoreactivity from human cerebral cortex and caudate nucleus revealed two major forms, one coeluting with sulphated cholecystokinin-8 and the other coeluting with sulphated cholecystokinin-58. Two minor components coeluting with cholecystokinin-4 and cholecystokinin-5 were also detected. The finding of cholecystokinin-like immunoreactivity corresponding to cholecystokinin-8 and cholecystokinin-58 in caudate nucleus where no preprocholecystokinin mRNA was found, indicates the presence of these peptides in afferent nerve terminals.

Microdialysis was combined with a highly sensitive sequential multiple antigen radioimmunoassay to simultaneously measure extracellular cholecystokinin and neurotensin fragments from discrete regions of the rat brain in vivo. The assay was conducted in 96-well plates and provided a limit of detection for both peptides of 0.1 fmol. Dialysis membranes composed of polyacrylonitrile, Cuprophan and polycarbonate were evaluated in vitro using both radiolabelled peptides and radioimmunoassay. Polycarbonate probes were implanted in the posterior medial nucleus accumbens-septum, medial caudate nucleus or medial prefrontal cortex of halothane-N2O-anaesthetized rats. Cholecystokinin immunoreactivity levels were generally above the assay detection limits (0.1-0.7 fmol) in 30-min samples from all three regions under basal conditions. Recovered basal amounts of neurotensin immunoreactivity were detectable in the nucleus accumbens-septum in approximately 50% of experiments (0.1-0.2 fmol) but were not measured in the caudate nucleus or prefrontal cortex. In the nucleus accumbens-septum, a 10-min pulse of 200 mM K(+)-containing artificial cerebrospinal fluid in the perfusion medium during a 30-min sampling period increased the recovered cholecystokinin and neurotensin immunoreactivity to 9.7 fmol +/- 1.9 S.E.M. and 5.8 +/- 1.6 S.E.M., respectively. A second stimulation following a 2.5-h interval produced similar elevations with S2:S1 ratios of 0.62 +/- 0.07 and 0.68 +/- 0.07 for cholecystokinin and neurotensin, respectively. In a separate series of experiments the second stimulation of both peptides was prevented by perfusion of a 10 mM EGTA-containing medium. Similar results were obtained in the caudate nucleus for cholecystokinin, but K(+)-induced elevations in neurotensin immunoreactivity were much smaller (0.5 fmol) in this brain region and calcium dependency was not established. Sequential K+ stimulations at 50, 100 and 200 mM produced progressively greater increases in recovered cholecystokinin and neurotensin immunoreactivity from the nucleus accumbens-septum and of cholecystokinin immunoreactivity from the prefrontal cortex. No neurotensin immunoreactivity was detected in the prefrontal cortex following K+ stimulation. Large post mortem increases in the recovered amounts of cholecystokinin and neurotensin immunoreactivity were observed. This effect was significantly attenuated by EGTA although there was a large calcium-independent component of the cholecystokinin immunoreactivity. On reverse-phase high-performance liquid chromatography the major cholecystokinin-immunoreactive peak co-eluted with sulphated cholecystokinin octapeptide. Neurotensin-immunoreactive material co-eluted with neurotensin (1-13), neurotensin (1-12), neurotensin (1-11), neurotensin (1-10) and neurotensin (1-8). These results further demonstrate the potential of microdialysis for studying neuropeptide release and metabolism in vivo when combined with sufficiently sensitive assay procedures.

The predominant immunoreactive cholecystokinin (CCK) forms in acid extracts of rat intestine eluted from reverse-phase high-performance liquid chromatography columns in the positions of CCK-8, CCK-33/39, and CCK-58. Control experiments indicated that smaller CCK forms did not arise from artifactual degradation of large CCK forms. Less than 10% of CCK-8 added to and extracted from intestine was recovered in acid; CCK-8 could not be recovered by subsequent alkaline extraction, but subsequent urea extraction yielded approximately 25% of the added peptide. This suggests that CCK binds to proteins during acid extraction and that the preponderance of large CCK forms in acid extracts is not due to inhibition of CCK degradation but results from poor extraction of small CCK forms. No evidence for a CCK-22-like peptide was found in acid or subsequent urea extracts of rat intestine, suggesting CCK posttranslational processing in adult rats is like that in humans and dogs.

The immunoreactivity of intact and trypsinized canine cholecystokinin-58 was examined using five C-terminally directed cholecystokinin antisera. Cholecystokinin-58 was nearly as immunoreactive as sulfated, amidated cholecystokinin-8 with one cholecystokinin-specific antiserum and an antiserum with moderate (18%) gastrin cross-reactivity, but two to four times less immunoreactive than the octapeptide with two other cholecystokinin-specific antisera and an antiserum with full cross-reactivity with cholecystokinin and gastrin. The cross-reactivity of cholecystokinin-58 with all antisera was increased by trypsinization, and the magnitude of the increase was related to the degree of trypsinization. These results suggest that it is not possible to measure absolute cholecystokinin levels in tissue and blood with antisera reported to date and that cholecystokinin-58 has a tertiary structure that influences its binding to cholecystokinin antibodies.

Molecular cloning of cholecystokinin (CCK) mRNA from porcine brain and gut has demonstrated that CCK is synthesized as an identical precursor in both tissues. The sequence for porcine CCK-58 predicted from CCK cDNA was identical with the amino acid sequence of the peptide purified from different lots of animals. However one group did report that there were differences in the N-terminus of CCK-58 purified from the intestines of two different lots of mongrel dogs. In the current report it is demonstrated that the amino acid sequences of CCK-58 purified separately from three bovine brains are identical through the first 19 N-terminal amino acid residues. The peptides were sequenced for ten additional steps and were shown to be identical with the previously reported sequences for the N-terminus of CCK-39. The N-terminus of bovine CCK-58 has the following sequence: AVPRVDDEPRAQLGALLAR.

The time-dependent release of molecular variants of cholecystokinin (CCK) into the circulation was studied before and 1, 2, and 4 h after a test meal in six healthy volunteers. At each time period, 100 ml of blood were drawn in a manner to inhibit CCK degradation. Plasma was formed and CCK concentrated by Sep-Pak C18 cartridge chromatography. Molecular variants of CCK and gastrin were well separated from each other by high-performance liquid chromatography (HPLC). Molecular forms of CCK and gastrin were measured by radioimmunoassay using an antibody that requires the presence of the carboxyl-terminal phenylalanine amide for full recognition, implying that biologically active forms were detected. HPLC elution positions of gastrin forms were determined using a gastrin-specific antibody. Chromatographic separation of CCK from gastrin forms was complete, allowing separate integration of gastrin and CCK forms. Therefore no subtraction of gastrin-like immunoreactivity from CCK-like immunoreactivity (CCK-LI) was necessary and CCK-LI could be directly determined. Peaks of CCK-LI were integrated in the column eluates and the plasma concentrations were calculated. Total plasma CCK-LI rose from a value of 2.4 +/- 0.6 pM before the test meal to 6.4 +/- 0.8, 6.6 +/- 0.9, and 5.8 +/- 1.2 pM 1, 2, and 4 h postprandially. The major molecular forms released into the circulation eluted on HPLC in the position of CCK-58 and CCK-39 (which coelutes with CCK-33). Minor amounts were detected in the position of CCK-8. There was no significant difference in the relative proportions of the molecular forms released at the different time periods.(ABSTRACT TRUNCATED AT 250 WORDS)

Analogs of cholecystokinin (Ac-CCK-7) in which Tyr(SO3H) was replaced with D-Tyr(SO3H), Tyr(m-SO3H), D-Tyr(m-SO3H) Tyr(o-SO3H) and D-Tyr(o-SO3H) were prepared by solid phase peptide synthesis, characterized by amino acid analysis, MS, UV, IR and tested in vivo for their ability to suppress food intake and in vitro (receptor binding). Comparison of the binding efficacy to food intake revealed a poor correlation. Ac-[Tyr(m-SO3H)]-CCK-7 retained substantial anorectic activity, whereas Ac-[Tyr(o-SO3H)-CCK-7 was essentially inactive. Ac-[D-Tyr(SO3H)]-CCK-7 and Ac-[D-Tyr(mSO3H)]-CCK-7 retained substantial biological activity. From these studies we conclude that the position of the negative charge on Tyr (meta or para) is important for anorectic activity, but the chirality of the alpha-carbon is not important for biological activity.

Total mRNA extracted from brain of a sea worm Nereis diversicolor (Annelida, Polychaeta) were injected into Xenopus oocytes. The in ovo-translated polypeptides were analyzed through electrophoresis of immunoprecipitated products or Western blotting techniques. Some of these polypeptides cross-reacted antibodies elaborated against three mammalian (human and rat) procholecystokinin (pro-CCK) sequences. Polypeptides of 15, 64, and 70 kDa were determined, but two families of less obvious products, whose molecular masses were between 27-34 kDa and 46-60 kDa, appeared. Furthermore, the Western blotting technique also showed a cross-reaction between some of the polypeptides (64 and 70 kDa) extracted from brains and the antibodies used. From this work, evidence is given of the presence of epitopes shared by cellular polypeptides extracted from the brain, in ovo-translated products and mammalian pro-CCK. Furthermore, these data suggest the existence of a CCK precursor in the brain of N. diversicolor.

Acid extracts of human intestines obtained from surgical samples or from organ donors contain cholecystokinin (CCK) immunoreactivity. From surgical samples, extracted and eluted quickly, greater than 75% of the CCK immunoreactivity eluted in the same region as purified canine CCK-58 during analytical reverse-phase high-pressure liquid chromatography (HPLC). A major portion of the CCK immunoreactivity from donor intestinal extracts also eluted in this region. This immunoreactivity has been purified from human intestinal extracts by a series of several reverse-phase and cation-exchange chromatographies. Amino acid and microsequence analysis showed that this immunoreactivity is human CCK-58. Tryptic digestion of purified human CCK-58 produced another immunoreactive form that eluted in the position of CCK-8 during analytical reverse-phase HPLC. The immunoreactivity of the trypsin-digested material was 2.6-fold higher than that of an identical sample of CCK-58 incubated without trypsin. Thus the carboxyl-terminal antibody used for radioimmunoassay cross-reacts greater than twofold less with human CCK-58. This diminished cross-reactivity would lead to an underestimation of the relative proportions of CCK-58 in tissue and plasma extracts. If CCK-58 is the major circulating form this diminished cross-reactivity would also lead to underestimations of the circulating levels of total CCK. Determination of human CCK-58 structure confirms that one of the major components of human CCK that expresses biological activity is CCK-58.

In this study we describe the synthesis and purification of biotinylated cholecystokinin-8 (Bio-CCK-8) and characterize its use as a probe for the pancreatic cholecystokinin receptor. CCK-8 (0.1 umoles) was reacted with either radiolabeled d-[8,9(-3)H]biotin succinimide ester (0.5 umoles) or N-hydroxysuccinimidyl-biotin in dimethylformamide and triethylamine, and purified by anion exchange chromatography. Concentrations of Bio-CCK-8 and CCK-8 needed for half-maximal inhibition of [125]I-CCK-8 binding to pancreatic membranes were the same (1.0 and 1.3 nM). Bio-CCK-8 retained full biological activity as determined by stimulation of pancreatic protein secretion from rats, and the biotin group bound to CCK-8 retained its high sensitivity for avidin.

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 affinity-chromatography 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.

To examine the distribution of cholecystokinins (CCKs) along the small intestine we examined the nature of CCKs in samples of jejunum, mid-intestine and ileum from human and porcine intestine. CCKs in intestinal mucosa were extracted by boiling in both neutral and acid conditions, and subjected to high pressure liquid chromatography (HPLC) to separate the forms of CCK followed by radioimmunoassay of separate fractions. In neutral extracts of human intestine CCK immunoreactivity totalled 119.4, 22.9 and less than 1 ng/g in jejunum, mid-intestine and ileum, whilst in acid extracts the corresponding values were 65.3, 47.4 and less than 1 ng/g. Amounts of CCK extracted from porcine mucosa were of similar magnitude. In neutral extracts material co-chromatographing on HPLC with synthetic porcine CCK 8 predominated, whilst in acid extracts material co-chromatographing with CCKs 33/39 was the major form. These forms of human and porcine CCKs extracted from the mucosa behaved similarly to CCK 8 and CCK 33/39 standards on HPLC, in the radioimmunoassay and on molecular exclusion chromatography - suggesting marked similarity of the CCKs in the two species. In both species there was a marked change in the ratios of CCK 8: CCK 33/39 down the intestine from 1: 0.8 in human jejunum to 1: 5.6 in mid-intestine and from 1: 1.5 in porcine jejunum to 1: 5.8 in mid-intestine. These observations may explain the changing patterns of CCKs in circulation with time after ingestion of a fat meal and the greater impairment of CCK 8 than CCK 33/39 release observed in coeliac disease.

In dispersed acini from guinea pig pancreas, cholecystokinin-(27-32)-amide [CCK-(27-32)-NH2] did not stimulate amylase secretion and inhibited the stimulation caused by CCK-(26-33). In dispersed acini from mouse or rat pancreas, CCK-(27-32)-NH2 stimulated amylase secretion. The stimulation of amylase secretion caused by a maximally effective concentration of CCK-(27-32)-NH2 was less than that caused by a maximally effective concentration of cholecystokinin-(26-33), and in contrast to the action of cholecystokinin-(26-33) supramaximal concentrations of CCK-(27-32)-NH2 did not cause submaximal stimulation of amylase secretion. Dibutyryl cGMP and proglumide each inhibited the stimulation of amylase secretion caused by CCK-(27-32)-NH2. CCK-(27-32)-NH2 inhibited binding of 125I-labeled CCK to mouse and rat pancreatic acini. These results indicate that, in mouse and rat pancreatic acini, CCK-(27-32)-NH2 is a partial agonist and that this partial agonist activity is produced by occupation of the CCK receptor by CCK-(27-32)-NH2.

This study describes a novel radioreceptor assay (RRA) for cholecystokinin (CCK) which is the first to measure and characterize brain CCK using a technique not dependent on the generation of peptide antibodies. The CCK RRA utilizes the mouse cerebral cortex CCK receptor as the binding source and [125I]BH-CCK-8 as the radiolabelled probe. [125I]BH-CCK-8 bound to the central CCK receptor with a Kd of 1.82 nM and a Bmax of 1.21 pmol/g tissue. Unlabelled CCK-8 displaced the specific binding of [125I]BH-CCK-8 with an inhibition constant of 3.84 nM. CCK was extracted (90% methanol) from discrete brain regions (mouse) and quantified using the CCK RRA. The amygdala contained the highest concentration of CCK (394 +/- 21 pmol/g tissue), followed by the olfactory bulbs (306 +/- 19 pmol/g tissue) and cerebral cortex (298 +/- 21 pmol/g tissue). Moderate levels of CCK were found in the hippocampus (212 +/- 18 pmol/g tissue), striatum (146 +/- 15 pmol/g tissue) and hypothalamus (129 +/- 9 pmol/g tissue). Low levels of CCK were recorded in the pons (45 +/- 5 pmol/g tissue), medulla (41 +/- 3 pmol/g tissue) and spinal cord (29 +/- 3 pmol/g tissue), whilst no CCK was detected in the cerebellum. The molecular forms of CCK in amygdala, cerebral cortex and hypothalamus were characterized using RRA in conjunction with HPLC. CCK-8 was identified as the major molecular form (88%, 94% and 91% of total CCK activity in amygdala, cortex and hypothalamus, respectively) with a smaller component attributable to CCK-4 (8%, 5% and 6% of the total CCK activity).

The primary structures of two novel forms of cholecystokinin, isolated from bovine upper intestine are reported. The two peptides are composed of 33 and 39 amino acid residues, respectively, the larger being an N-terminally extended form of the shorter peptide. The primary structure of the 39 amino acid peptide is: (Formula: see text) This amino acid sequence differs from the porcine hormone at positions 13 and 15, which are Val and Met, respectively, in pig, the same amino acid substitutions have previously been found to occur also in dog.

Most preparations of cholecystokinin reported to release insulin have been impure. When highly purified preparations of extracted cholecystokinin and also the synthetic C-terminal octapeptide of the hormone became available for use in humans, we investigated their insulinotropic activity in comparison with a cruder preparation of cholecystokinin in 10 fasting non-diabetic subjects. The doses employed were 75 Ivy dog units, except for the synthetic C-terminal octapeptide of cholecystokinin that was given in a dose of 200 Ivy dog units to compensate for a shorter hawn every minute during 10 min after each injection, thereafter at intervals of 5 min. The mean plasma insulin level increased significantly, reaching a peak 4-5 min after iv injection of the cruder cholecystokinin preparation, but after the other two preparations the plasma insulin level was not significantly changed. The blood glucose level was not significantly changed by any of the preparations used. It is concluded that the plasma insulin peak seen in man after i.v. injection of the less highly purified preparation was due not to cholecystokinin but to some other agent present in this less pure preparation. The identity of this factor is discussed.