Degradation of cholecystokinin-like peptides by a crude rat brain synaptosomal fraction: a study by high pressure liquid chromatography

Degradation of CCK-8, CCK-4, and related peptides by a crude synaptosomal fraction of rat brain was investigated by monitoring the tryptophan fluorescence of reaction products after HPLC fractionation. At 20 degrees C, the half disappearance time was 52 min for CCK-8, 35 min for unsulphated CCK-8, 20 min for unsulphated CCK-7, 6 min for Tyr(SO3H)-Trp-Met-Asp-Phe-NH2, and 3 min only for CCK-4. Caerulein was much more resistant than CCK-8, and Boc-CCK-4 and Aoc-CCK-4 remained stable for at least 3 h. The apparent Km for CCK-8 and CCK-4 was 40 microM and maximal activity on CCK-8 was observed at pH 7.0. Zn2+ was strongly inhibitory. The protease inhibitors puromycin and bacitracin, the metal chelator 1,10-phenanthroline, and the sulphydryl blocking agents N-ethylmaleimide and p-chloromercuribenzoate greatly reduced the release of tryptophan from CCK-8. Puromycin inhibition of CCK-8 degradation provoked the accumulation of a CCK-7-like peptide, and that of CCK-4 degradation was of a competitive type (Ki = 2 microM). The CCK-8 degrading activity of brain synaptosomes was present in the cytosol as well as in synaptic membranes.     

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

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

Degradation of human gastrin and CCK by endopeptidase 24.11: differential behaviour of the sulphated and unsulphated peptides

The degradation of human sulphated heptadecapeptide gastrin (G17s) by human endopeptidase 24.11 was studied in vitro. The products of degradation were characterized by HPLC, region-specific gastrin radioimmunoassay and amino acid analysis. The enzyme cleaved G17s at four sites, Trp4-Leu5, Ala11-Tyr12, Gly13-Trp14 and Asp16-Phe17. The patterns of fragments produced when sulphated and unsulphated G17s are hydrolysed by endopeptidase 24.11 indicate that the enzyme cleaves both substrates at the same four bonds. However, the sulphated G17 was 3-times less rapidly degraded than the unsulphated G17 (G17ns). In contrast, the rate of cleavage of the octapeptide cholecystokinin (CCK8) was faster when the peptide was sulphated. The kinetic data of endopeptidase 24.11 indicated similar Km values for sulphated or unsulphated gastrin and CCK; sulphated CCK8 exhibited a 2-fold higher kcat/Km value compared to unsulphated CCK8, whereas G17s exhibited a 2-fold lower kcat/Km value compared to G17ns. The results indicate that the presence of a sulphate group causes a marked reduction in the rate of hydrolysis of gastrin by endopeptidase 24.11, whereas sulphation enhances cholecystokinin degradation by the same enzyme. They also suggest that endopeptidase 24.11 may be responsible for the difference in metabolism of sulphated and unsulphated G17, previously observed in human circulation.     

Measurement of cholecystokinin octapeptide using a new specific radioimmunoassay

A peptide analogue of CCK-8 (Tyroc) which has a tyrosine in place of the amide group in the C-terminal end, has been used both for raising antisera and for iodination. The antisera produced by immunisation with Tyroc are directed towards the N-terminal end of the CCK-8 molecule. The assay system appears totally specific for the CCK-8 sulphated molecule and shows no significant cross-reaction with other molecular forms of CCK, or with the gastrins. The assay can detect changes between adjacent tubes of 0.25 fmol/tube CCK-8 with 95% confidence. The assay is robust, reliable and reproducible and can be used to measure tissue and plasma levels of CCK-8.     

Cholecystokinin octa- and tetrapeptide degradation by synaptic membranes. II. Solubilization and separation of membrane-bound CCK-8 cleaving enzymes

Solubilization of rat synaptic membranes by Triton X-100, followed by DEAE-cellulose chromatography allowed the identification of different CCK-8 cleaving enzymes. The first one (in the order of elution) removed the N-terminal aspartic acid residue of CCK-8 and was active on L-aspartic acid beta naphtylamide, suggesting that a corresponded to an aminopeptidase A. Two aminopeptidases of broad specificity hydrolyzed sequentially all the peptide bonds of CCK-8 as far as the release of free tryptophan. The removal of the sulfated tyrosine residue of CCK-8 occurred at a slower rate than that of the unsulfated residue. Another peptidase converted CCK-8 into its C-terminal heptapeptide. This enzyme had a lower affinity for the sulfated octapeptide in comparison with the unsulfated form (app Km of respectively 180 and 40 muM). The CCK-7 generating proteases displayed a moderate regional variation in five rat brain areas, with the highest activity in olfactory bulbs membranes and the lowest in cerebellar membranes. This distribution followed (with a lower amplitude) that of the CCK receptors.     

Cholecystokinin octapeptide analogues stable to brain proteolysis

Based on recent findings identifying the initial degradative cleavage of CCK-8 at the Met3-Gly4 bond by a metalloendopeptidase, two analogues of CCK-8 with D-Ala and D-Trp substitutions at the Gly4 position were synthesized as stable analogues. Their stability to proteolysis by brain membranes and their binding potency at central CCK receptors were quantified. Both peptides are stable to degradation by peptidases in cortical synaptic membrane preparations. The analogues are nearly equipotent to CCK-8 in their affinities for inhibition of 125I-CCK-33 binding to guinea pig cortical membranes. L-Ala and L-Trp substituted peptides were synthesized for comparison. Both these peptides are degraded by synaptic membranes and the L-Trp substituted peptide possesses a greatly reduced affinity for central CCK receptors. Therefore, the structure of CCK due to the D conformation of Gly is more capable of interacting with brain CCK receptors. Further conformational analysis will establish whether the stabilized structure is a beta-bend or a beta-turn. Since these peptides are highly potent and stable to brain proteolysis they may be useful as stable CCK analogues for in vivo application.     

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

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

Intestinal metabolism and absorption of cholecystokinin analogs in rats

Intestinal metabolism and poor permeability were known to be major barriers for oral absorption of large peptide drugs. Dimensionless wall permeability values of C-terminal octa- and tetra-peptides cholecystokinin analogs (CCK8 and CCK4) were estimated and found out to be greater than 1, suggesting no permeability-limited absorption for CCK analogs. Thus, a strategy employing enzyme inhibitors and a specific delivery site to improve the absorption was developed and tested with CCK8, followed by identification of metabolites of the analogs and their participating enzymes in rabbit brush-border membrane vesicles. Thiorphan and amastatin, a specific enzyme inhibitor for enkephalinase and aminopeptidase, respectively, in pH 4 buffer solution were coadministered with CCK8 to the ileum in fistulated rats. The absolute bioavailability (F) of CCK8 was 5.4% and increased to 19% in the presence of the enzyme inhibitors, while the F values following oral administration were close to zero. These results indicate that peptide oral delivery is possible.     

Receptor binding of cholecystokinin analogues in isolated rat pancreatic acini

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

缩胆囊素受体拮抗剂对血浆缩胆囊素生物测定的影响

利用生物法测定了102例正常人空腹及进食二个油煎鸡蛋30min后血浆CCK浓度,结果分别为1.4±0.1pmol/L和4.9±0.4pmol/L(X±SE)。血浆CCK浓度不随性别和年龄而变化。本文研究了三种不同类型的CCK受体拮抗剂对CCK生物测定的影响。一组将L364,718(5.0nmol/L),丙谷胺(1.0mmol/L)或Bt_2-cGMP(0.1mmol/L)分别加入含8pmol/LCCK-8的人血浆,用SEP-PAK提取,另一组先提取血浆,于测定前向血浆提取物内加入上述拮抗剂,两组同时测定。结果显示,在有L364,718存在时仍可利用生物法测定血浆CCK浓度,如血中含有丙谷胺或Bt_2-cGMP则无法准确测定。     

Possible degradative process of cholecystokinin analogs in rabbit jejunum brush-border membrane vesicles

Our recent work on the intestinal metabolism and absorption of cholecystokinin analogs, sulfated C-terminal octapeptide (CCK8; Asp-Tyr(SO(3)H)-Met-Gly-Trp-Met-Asp-Phe(NH(2)) = DY(SO(3)H)MGWMDF(NH(2))) and tetrapeptide (CCK4; Trp-Met-Asp-Phe(NH(2)) = WMDF(NH(2))), was extended to investigate the degradative process of these analogs using rabbit jejunum brush-border membrane vesicles and to find a better enzyme-inhibitor system for intestinal absorption of peptide drugs. Various enzyme inhibitors and a lower pH buffer were applied to discover the major enzyme(s) involved in each process. Metabolic pathways showing degradative processes were proposed for both analogs. The major cleavage site occurs at the W(1)-M(2) for CCK4. At least three metabolic pathways occur independently for CCK8 and appear at peptides bonds between G(4)-W(5), M(6)-D(7), and D(7)-F(NH(2))(8). Many different enzymes of aminopeptidase, endopeptidase, angiotensin-converting enzyme, metalloenzyme, and others were involved in each process. Identification of more specific yet safe enzyme inhibitors and co-administration of various these inhibitors may lead to further enhancement in intestinal peptide absorption when administered orally.     

Aminopeptidase resistant Arg-Gly-Asp analogs are stable in plasma and inhibit platelet aggregation.

Tetrapeptides containing the sequence Arg-Gly-Asp (RGD) antagonize fibrinogen binding to its platelet receptor (gp IIb/IIIa, integrin alpha IIb beta 3) and inhibit platelet aggregation in vitro. The peptides RGDS and RGDY(Me)-NH2 were rapidly degraded when incubated in human, rat, and dog plasma. HPLC analysis indicated that amino acids were sequentially removed from the peptide N-terminus, and this degradation was prevented by the aminopeptidase inhibitor bestatin. Analogs of RGDY(Me)-NH2 with an acetylated or deleted alpha-amino group were prepared. Both analogs were stable when incubated in plasma, blocked 125I-fibrinogen binding to activated platelets (IC50 = 10-30 microM) and inhibited ADP induced platelet aggregation (IC50 = 10-30 microM). This study concludes that aminopeptidase rapidly degrades RGD peptides in plasma, an important issue for in vivo testing of RGD peptides and analogs. RGD analogs intrinsically stabilized against aminopeptidase are stable in plasma and are important tools for antithrombotic studies involving antagonism of gp IIb/IIIa.     

In vitro transport of an allatostatin across the foregut of Manduca sexta larvae and metabolism by the gut and hemolymph

The degradation of synthetic cydiastatin 4 by enzymes of the foregut and hemolymph, and transport across the foregut of larvae of the tobacco hawkmoth moth, Manduca sexta, were investigated using reversed-phase high performance liquid chromatography (RP-HPLC) together with matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). In the hemolymph in vitro, cydiastatin 4 had a half-life of ca. 30 min. Two degradation products were identified; cydiastatin 4(1-6), due to cleavage of the C-terminal di-peptide GL-amide, and cydiastatin 4(2-8), due to cleavage of the N-terminal A residue. This hydrolysis could be inhibited by up to 93% by 1,10-phenanthroline. Other protease inhibitors had lesser effects (<21% inhibition of degradation) including the aminopeptidase inhibitors amastatin and bestatin, and the chelator EDTA. When incubated with foregut extract in vitro, cydiastatin 4 had a half-life of 23 min, and the hydrolysis products detected were also cydiastatin 4(1-6) and cydiastatin 4(2-8). Similarly, 1-10 phenanthroline inhibited foregut enzyme degradation of cydiastatin 4 by ca. 80%, whereas amastatin, bestatin, and EDTA had very little effect (<10% inhibition). Cydiastatin 4 was transported, intact, from the lumen to the hemolymph side of foregut tissues that were mounted as flat sheets in modified Ussing chambers. This trans-epithelial flux of peptide was dose and time-dependent, but was <3% of the amount of cydiastatin 4 present in the lumen bathing saline. In contrast, no trans-epithelial transport of peptide was apparent across everted foregut sac preparations.     

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.     

Degradation of thymic humoral factor γ2 in human,rat and mouse blood: An experimental and theoretical study

The degradation of the immunomodulatory octapeptide, thymic humoral factor γ2 (THF-γ2, thymoctonan) has been studied in whole blood samples from human, rat and mouse. The peptide, Leu-Glu-Asp-Gly-Pro-Lys-Phe-Leu, was shown to be rapidly degraded by peptidases. The half-life of the intact peptide was less than 6 min at 37 °C in blood from the three species tested. The main fragments formed from THF-γ2 were found to be Glu-Asp-Gly-Pro-Lys-Phe-Leu (2–8), Asp-Gly-Pro-Lys-Phe-Leu (3–8) and Glu-Asp-Gly-Pro-Lys (2–6) in human and in rat blood and 2–8 and 2–6 in mouse blood. Analysis of the time course of degradation revealed a sequential removal of single amino acids from the N-terminus (aminopeptidase activities) in a process that was apparently unable to cleave the Gly-Pro bond (positions 4–5 in the peptide) together with an independent cleavage of the Lys-Phe bond (positions 6–7 in the peptide) to release the dipeptide Phe-Leu. This behaviour and the effects of inhibitors showed the involvement of metallo-exopeptidases in the N-terminal digestion and a phosphoramidon-sensitive metallo-endopeptidase in the cleavage of the Lys-Phe bond. The degradation patterns in human blood were modelled in terms of the competing pathways involved approximating to first-order kinetics, and an analytical solution obtained via the method of Laplace Transforms. The half-life of THF degradation in whole rat blood sample was found to be significantly lower than in human or mouse.     

Cholecystokinin Modulates the Release of Dopamine from the Anterior and Posterior Nucleus Accumbens by Two Different Mechanisms

The effects of various cholecystokinin (CCK)-related peptides were investigated on 35 mM K(+)-stimulated endogenous dopamine release from slices of either anterior or posterior nucleus accumbens of the rat. CCK sulphated octapeptide (1-10 microM), but not pentagastrin or CCK unsulphated octapeptide, was found to cause a dose-dependent increase in the release from the posterior nucleus accumbens. This effect was blocked by low doses of the CCKA receptor antagonist L364,718 (10 nM) but not the CCKB receptor antagonist L365,260. In the anterior nucleus accumbens CCK sulphated octapeptide (1 microM) and CCK unsulphated octapeptide (0.1-1 microM) inhibited the dopamine release, and this effect was blocked by L365,260 (10-100 nM) but not by L364,718. These results suggest that CCK has a different effect on dopamine release from the anterior and posterior nucleus accumbens and that these effects are mediated by two different types of CCK receptor.     

The short term satiety peptide cholecystokinin reduces meal size and prolongs intermeal interval

Camostat mesilate (or mesylate) releases endogenous cholecystokinin (CCK) or CCK-58, the only detectable endocrine form of CCK in the rat, and reduces cumulative food intake by activating CCK₁ receptor. However, the literature lacks meal pattern analysis and an appropriate dose-response curve for this peptide. Therefore, the current study determines meal size (MS), intermeal interval (IMI) and satiety ratio (SR) by orogastric gavage of camostat (0, 12.5, 25, 50, 100, 200, 300, 400, 800 mg/kg) and compares them to those previously reported by a single dose of CCK-8 (1 nmol/kg, i.p), the most utilized form of CCK. We found that camostat (200, 300, 400 and 800 mg/kg) and CCK-8 reduced cumulative food intake and the size of the first meal, but only camostat prolonged IMI and increased SR. There was no change in the duration of the first two meals or in rated behaviors such as feeding, grooming, standing and resting in response to camostat and CCK-8, but there was more resting during the IMI in response to camostat. This study provides meal pattern analysis and an appropriate dose-response curve for camostat and CCK-8. Camostat reduces food intake by decreasing MS and prolonging IMI, whereas CCK-8 reduces food intake by reducing only meal size.     

Alkaline extraction of cholecystokinin-immunoreactivity from rat brain

Alkaline aqueous extractants remove from rat brain 2 to 4 times the CCK-immunoreactivity that is removed by acidic or neutral aqueous extractants. The distribution among the various hormonal forms appears to be independent of the extractant: about $\text{1}\text{10}$ in the larger basic forms (CCK-33 and CCK-39); about $\text{1}\text{4}$ as the C-terminal dodecapeptide (CCK-12) and the remainder as the octapeptide (CCK-8). In contrast, alkaline and acidic aqueous solutions are equally efficient in extraction of enkephalin-immunoreactivity from the same tissues. We are presently unable to account for the very different efficiencies of the various extractants in removing CCK-immunoreactivity from brain.     

Hydrolysis of the C-terminal octapeptide of cholecystokinin by rat kidney membranes: characterization of the cleavage by solubilized endopeptidase-24.11

Rat kidney membranes were solubilized by Triton X-100 and the CCK-8 degrading peptidases were resolved by chromatography on DEAE-cellulose. Four proteases were detected: two phosphoramidon-sensitive endopeptidases (EC 3.4.24.11), a bestatin-sensitive aminopeptidase and an unidentified enzyme. The pattern of cleavage of CCK-8 and shorter C-terminal fragments by endopeptidase 24.11 was investigated and indicated that the Gly29-Trp30, Trp30-Met31 and Asp32-Phe33 were scissile bonds. However, the cleavage pattern differed markedly from one CCK peptide to another: in the penta- and hexapeptide of CCK the bonds hydrolyzed were either Asp-Phe and Trp-Met or, Asp-Phe and Gly-Trp, respectively. The presence of the sulfate group on the tyrosine residue of CCK-8 influence markedly the nature of the major cleavage fragments produced by the endopeptidase. The major bonds cleaved were Asp-Phe, Trp-Met and Gly-Trp for unsulfated CCK-8, whilst for the sulfated octapeptide, the Trp-Met bond became a minor cleavage site.     

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.