C-terminal Cholecystokinin Research Articles

Artificial receptor for peptide recognition in protic media: The role of metal ion coordination

The production of molecularly imprinted polymers (MIPs) for the recognition of C-terminal cholecystokinin pentapeptide (CCK-5) in the presence of metal ion is reported. The MIPs were produced under the same molar ratio of template to monomers (acrylamide, N, N′-methylene bisacrylamide) in the presence or absence of nitrilotriacetic acid–nickel (Ni–NTA) complex. Scanning electron microscopy images of MIPs were obtained in an attempt to correlate the adsorption characteristics with polymer's morphology. Subsequently Ni 2+ was removed and substituted by other divalent ions such as Mg 2+, Fe 2+, Zn 2+, Co 2+ and Cu 2+. It was found that polymers containing the metal ion complex with the order Fe–NTA, Ni–NTA and Cu–NTA presented lower dissociation constant values than the rest thus exhibiting stronger guest binding activity. The percentage of theoretical maximum binding sites B max was almost the same for these ions, indicating that the ion-template coordination is responsible only for their binding strength and not for the number of active sites. The data showed that the proper ion or the ion exchange in the produced MIPs could lead to materials with interesting recognition abilities regarding CCK-5 or/and other isoforms of CCK hormone.

Evaluation of Contrast-Enhanced Ultrasound of the Pancreas Combined with Concurrent Hormone Stimulation

To evaluate the potential of combined administration of the gastrointestinal hormones secretin (Secrelux) and c-terminal cholecystokinin (CCK-8 s) together with contrast-enhanced ultrasound (CE-US) to generate an extended contrast enhancement of healthy pancreatic tissue. 14 anaesthetised pigs weighing 30-35 kg were studied. After laparotomy, the pancreas was located and a B-mode examination followed by a CE-US examination of the gland was made using SonoVue 1.5 ml. After an injection of Secrelux 1 U/kg and CCK-8 s 100 pmol/kg, a second CE-US examination was conducted. The hormones and the contrast agent were administered through a catheter in the superior vena cava. The sonographic images were stored for later evaluation. The study showed that CE-US increased the echogenicity of the pancreas by an average of 15.6 decibel (dB) (confidence intervals [CI]: 13.72, 17.42) p < 0.0001, an increase of 24%. The administration of Secrelux and CCK-8 s in combination with CE-US further increased the echogenicity of the pancreas by an average of 3% (CI: 0.36, 5.36) p = 0.028. A new sequence of hormones and CE-US 20 min after the previous injection did not induce further enhancement. The area under the curve (AUC) was significantly larger using both hormones and CE-US compared with CE-US alone by an average of 66 dBx sec (CI: 28,103) p = 0.002. It is possible to generate an extended contrast enhancement of healthy pancreatic tissue using CE-US combined with the administration of the gastrointestinal hormones secretin (Secrelux) and c-terminal cholecystokinin (CCK-8 s). Our results may improve the ability to discriminate between healthy pancreatic tissue and areas with a changed blood flow due to either neoplasm or other pathological lesions.

Physicochemical properties of mixed micellar aggregates containing CCK peptides and Gd complexes designed as tumor specific contrast agents in MRI.

New amphiphilic molecules containing a bioactive peptide or a claw moiety have been prepared in order to obtain mixed micelles as target-specific contrast agents in magnetic resonance imaging. The first molecule, C(18)H(37)CONH(AdOO)(2)-G-CCK8 (C18CCK8), contains a C18 hydrophobic moiety bound to the C-terminal cholecystokinin octapeptide amide (CCK 26-33 or CCK8). The second amphiphilic compound, C(18)H(37)CONHLys(DTPAGlu)CONH(2) (C18DTPAGlu) or its gadolinium complex, (C18DTPAGlu(Gd)), contains the same C18 hydrophobic moiety bound, through a lysine residue, to the DTPAGlu chelating agent. The mixed aggregates as well as the pure C18DTPAGlu aggregate, in the presence and absence of Gd, have been fully characterized by surface tension measurements, FT-PGSE-NMR, fluorescence quenching, and small-angle neutron scattering measurements. The structural characterization of the mixed aggregates C18DTPAGlu(Gd)-C18CCK8 indicates a spherical arrangement of the micelles with an external shell of approximately 21 A and an inner core of approximately 20 A. Both the DTPAGlu(Gd) complexes and the CCK8 peptides point toward the external surface. The measured values for relaxivity in saline medium at 20 MHz proton Larmor frequency and 25 degrees C are 18.7 mM(-)(1) s(-)(1). These values show a large enhancement in comparison with the isolated DTPAGlu(Gd) complex.

Mimicry of beta II'-turns of proteins in cyclic pentapeptides with one and without D-amino acids.

The solution structure of eight cyclic pentapeptides has been determined by two-dimensional 1H-NMR spectroscopy combined with spectra simulations and restrained molecular dynamic simulations. Six of the cyclic pentapeptides were derived from the C-terminal cholecystokinin fragment CCK-4 enlarged with Asp1 resulting in the sequence (Asp-Trp-Met-Asp-Phe), one L-amino acid after the other was substituted by its D-analog. In addition, two peptides, including an all-L-amino-acid-containing cyclic pentapeptide, cyclo(Asp-Phe-Lys-Ala-Thr) and cyclo(Asp-Phe-Lys-Ala-D-Thr) were investigated. All D-amino-acid-containing peptides show beta II'-turn conformations with the D-amino acid in the i + 1 position, excepting the D-aspartic-acid-containing peptides. These two peptides are characterized by the lack of beta-turns at pH values less than 4, suggesting that D-aspartic acid in the full-protonized state avoids the formation of beta-turns in these compounds. At pH values greater than 5, a conformational change into the beta II'-turn conformation was also observed for these peptides. Conformations without beta-turns are expected for cyclic all-L pentapeptides, but both cyclo(Asp-Phe-Lys-Ala-Thr) and the D-Thr analog cyclo(Asp-Phe-Lys-Ala-D-Thr) exhibit beta II'-turn conformations around Thr-Asp and D-Thr-Asp. Thus cyclic all-L pentapeptides and those with one D-amino acid are able to form similar structures preferably with a beta II'-turn. The beta-turn formation in cyclic pentapeptides containing a D-aspartic acid is dependent on the ionization state. The relevance of the work to the design of beta'-turn mimetics is discussed.

Full and partial agonist activity of C-terminal cholecystokinin peptides at the cloned human CCK-A receptor expressed in Chinese hamster ovary cells.

The agonist activities of the C-terminal cholecystokinin peptides sulfated cholecystokinin octapeptide (CCK-8S), non-sulfated cholecystokinin octapeptide (CCK-8NS), pentagastrin and CCK-4 at the cloned human CCK-A receptor expressed in Chinese hamster ovary cells were evaluated in two functional assays of receptor activation. [125I]-CCK-8S displacement studies employing membranes derived from these cells revealed the expected rank order of affinity for a number of CCK receptor ligands. CCK-8S was a potent agonist in (i) stimulating the mobilization of intracellular free Ca2+, measured with the Ca2+ sensitive fluorescent indicator FURA-2, and (ii) stimulating increases in extracellular acidification rates, measured by microphysiometry. Consistent with their lower affinities for CCK-A receptors, CCK-8NS, pentagastrin and CCK-4 were weaker agonists in both functional assays. In addition, these peptides exhibited partial agonist activity relative to the maximum response observed with CCK-8S in both assays. These results demonstrate that CCK-8S represents the minimum ligand requirement for both high affinity and full agonist activity at the human CCK-A receptor subtype.

Induction and Detection of Anti-Peptide Antibody Specificity Is Critically Affected by the Mode of Hapten Presentation

C-Terminal cholecystokinin (CCK)-peptides of increasing chain lengths were all linked at their N-termini to the single surface-exposed cysteine residue 107 of yeast iso-1-cytochrome c by the maleimide/thiol reaction. The resulting CCK/cytochrome 1:1 conjugates with the haptenic peptides in the identical protein environment were used to immunize outbred guinea pigs in order to assess the critical size of CCK peptides required for the expression of a CCK-specific epitope and the induction of antibodies not crossreacting with the homologous gastrin sequence. By using standard ELISA techniques with polystyrene-adsorbed antigen to evaluate the specificity of the antisera, none of the conjugates were found to induce anti-CCK antisera not crossreacting with gastrin. However, when the biotinyl-CCK-antigen was immobilized by polystyrene-adsorbed avidin, i.e. via a procedure which assures maximum accessibility of the bound antigen, we were able to demonstrate that with CCK-12 and particularly CCK-13, linked through their N-termini to the carrier, the critical length for the expression and recognition of a CCK-specific epitope was reached. The related polyclonal antisera did not crossreact with the homologous gastrin in the modified ELISA.

Receptor selectivity of cholecystokinin effects on mesoaccumbens dopamine neurons

Extracellular recording techniques were combined with antidromic stimulation to examine the effects of C-terminal cholecystokinin (CCK) fragments and CCK antagonists on the activity of identified mesoaccumbens dopamine (MADA) neurons in chloral hydrate-anesthetized rats. These experiments were designed to determine the receptor selectivity of sulfated CCK octapeptide (CCK-8S) effects on MADA cells. Neither CCK tetrapeptide (CCK-4) nor unsulfated CCK octapeptide (CCK-8U) significantly altered MADA cell basal firing rate or responsiveness to the inhibitory effects of the D2 DA agonist quinpirole. As reported previously for ventral tegmental area DA cells, CCK-8S produced increases or decreases in the firing rate of most MADA cells sampled. CCK-8S also enhanced the sensitivity of MADA neurons to quinpirole-induced inhibition. This increase in sensitivity to quinpirole was blocked by pretreatment with the nonselective CCK receptor antagonist proglumide and the preferential CCK-A receptor antagonist CR 1409 but not by the preferential CCK-B receptor antagonist L-365,260. The inactivity of CCK-4 and CCK-8U in these tests and the results with the antagonists suggest that the effects of CCK-8S on MADA neuronal activity are mediated by CCK-A receptors.

Ultrastructure of E. coli: An in vitro study of cells cultured in the presence of four gastrointestinal (GI) hormones

Hormones are found in exocrine secretions entering the gut. They alter the morphology of many eukaryotic cells; whether they affect the morphology of enteric flora is unknown. In this study, we examined the ultrastructure of E. coli, a common bacterium in the mammalian gut, for morphological changes resulting from exposure to GI hormones.E. coli (#11775 from American Type Culture Collection) were grown in protease-free trypticase soy broth (TSB) at 37°C for 18 hr to a concentration of 2 x 107 cells/ml. Pure synthetic hormones were used: sulfated C-terminal cholecystokinin octapeptide (CCK), pentagastrin (PG), cyclic somatostatin tetradecapeptide (SS), or the porcine form of secretin (SEC). These were individually added to. bacterial cultures in TSB to make 1 x 107 organisms/ml and 0.0, 0.5, 2.5, or 5.0 μg of hormone/ml, then incubated for 30 min at 37°C. The cultures were rapidly chilled and added to equal volumes of cold 6% glutaraldehyde in 0.2 M cacodylate buffer. After 30 min, the bacteria were concentrated by centrifugation (15 min at 4000 RPM) and the pellets suspended in cold 3% glutaraldehyde for an additional 15 min, followed by centrifugation. The pellets were resuspended in cold cacodylate buffer and stored at 2°C for 1-7 d. The cells were again centrifuged and the pellets were blotted with a strip of filter paper to remove excess fluid, then mixed with a drop of warm 2% agar. The agar suspensions were pipetted into cold saline. The resulting solidified extrusions were cut by hand into 2 mm segments for further processing in 1% OsO4 (with or without en bloc staining in 2% uranyl acetate (UA) in ethanol). Following dehydration in ethanol, rinsing in propylene oxide, and encapsulation in Epon-Araldite, thin sections were examined and photographed with a JEOL-100C microscope.

Potentiation of β-endorphin effects by cholecystokinin antiserum in rats

Intracerebroventricular administration of a C-terminal cholecystokinin (CCK) antiserum potentiated the analgesic and cataleptic effects of beta-endorphin. The results are opposite to those observed after injection of CCK-8. It was suggested that CCK-8 may play a physiological role antagonizing the action of beta-endorphin.

A new class of potent gastrin antagonists

The hexapeptide Z-Tyr(SO 3 −)-Met-Gly-Trp-Met-Asp-NH 2, from the natural sequence of C-terminal cholecystokinin was found to be a competitive antagonist of cholecystokinin receptors, in vitro. In the present study, we report that this peptide inhibits gastrin-induced acid secretion in vivo, (ED 50 = 1.5 μmol · kg −1), without agonist activity. Desulfation of the tyrosine residue slightly altered this effect. The tripeptide Boc-Trp-Met-Asp-NH 2 showed similar effects, but had lower potency (ED 50 = 12 μmol · kg −1). From these preliminary results, it can be concluded that removal of the phenylalanine residue from the C-terminal sequence of CCK or gastrin, leads to an antagonist of the natural hormones and that C-terminal phenylalanine residue is important for agonist activity. As compared with proglumide, a well known gastrin receptor antagonist, these peptides were 20–200 times more potent as inhibitors on the same model.