Degradation of cholecystokinin octapeptide,related fragments and analogs by human and rat plasma in vitro

The rate of degradation of cholecystokinin octapeptide, related fragments and analogs by human and rat plasma was investigated, using high pressure liquid chromatography for the separation and identification of the degradation products.CCK tetrapeptide showed a half-life of 13 min in human plasma while its cleavage in rat plasma occurred at a very high rate (half-life of less than 1 min).The kinetics of disappearance of both sulphated and unsulphated CCK-8 indicated more than a single rate of degradation; the faster degrading system showed a half-life of 18 min for unsulphated CCK-8 and of 50 min for the sulphated peptide in human plasma as compared respectively with 5 and 17 min in rat plasma. The cleavage of CCK-8 was inhibited by bestatin and puromycin, suggesting that aminopeptidases play a major role in the breakdown of this peptide.CCK-9 analogs were rapidly converted into their corresponding octapeptides (half-life of 2.7 min in human plasma). This conversion was inhibited by puromycin and bestatin, suggesting the participation of aminopeptidase(s) probably specific for basic amino acids.CCK decapeptide exhibited a greater stability than the nonapeptides (half-life of 30 and 45 min in human and rat plasma respectively) and also gave rise to CCK-8 as degradation product. This cleavage was not affected by aminopeptidase inhibitors but was decreased by aprotinin (Trasylol®), suggesting that trypsin-like and/or kallikrein-like enzyme(s) were involved in the plasma metabolism of this peptide.     

Postnatal development of the cholecystokinin-gastrin family of peptides in the brain and gut of rat

The average levels of CCK-8- and CCK-like peptides present in aqueous and acidic extracts of the brain of the newborn rat were, respectively, 5.3 and 2.9 pmol/g wet weight. These low levels increased 20- and 10-fold, respectively, so as to attain, at 25–30 days post partum, values comparable to those found in adult brain. There was also a rapid deposition of CCK-gastrin-like peptides in the whole gut between day 20 post coitum and day 10 post partum, when maximal concentrations were attained. Later, less CCK-gastrin peptides than other gut components were accumulated during the progressive weaning of the young rats, so that the average concentrations of CCK-gastrin-like peptides were, at day 30, similar to those observed in adult gut.     

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.     

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.     

Cholecystokinin octa- and tetrapeptide degradation by synaptic membranes. III. Inactivation of CCK-8 by a phosphoramidon-sensitive endopeptidase

Solubilization of rat synaptic membranes by Triton X-100 followed by DEAE-cellulose chromatography allowed the separation of a phosphoramidon-sensitive endopeptidase that cleaved CCK-8. This enzymatic activity revealed similar if not identical to "enkephalinase A." A major cleavage point, at the Trp30-Met31 bond, and a minor one at the Tyr27-Met28 bond were identified in the sequence of CCK-8. Replacements of the Met28 and Met31 residues by Thr and either Leu or Nle respectively, in CCK-9 analogues, did not improve the resistance of these peptides to enzymatic degradation. The regional distribution in rat brain of this CCK-8 cleaving endopeptidase displayed marked variations with the highest activity in striatal membranes; it closely followed that described for "enkephalinase" in mouse brain.     

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.     

Cerebral ventricular transport and uptake: Importance for CCK-mediated satiety

Brain cholecystokinin (CCK) peptides have been proposed to be involved in the control of feed intake. We have examined the importance of the cerebral ventricular system in CCK-mediated satiety in sheep. Continuous injection of 0.64 pmol/min CCK-8 into the lateral ventricles (LV) decreased feeding, whereas injection of neither 0.64 nor 2.55 pmol/min CCK-8 into the cisterna magna (CM) significantly affected feeding. Thus, it is likely that the rostral, but not caudal, ventricular compartments and/or adjacent brain areas are involved in CCK-8 mediated satiety. The rate of injection of carrier solution (synthetic cerebrospinal fluid [sCSF]) was found to affect feed intake during a continuous 75 min injection: feed intakes were greater during injection of sCSF at 0.10 ml/min than during either 0.03 ml/min sCSF or no injection (sham). Injection of 0.64 pmol/min CCK-8 in either 0.03 or 0.10 ml/min decreased feeding. The increased feeding during 0.10 ml/min sCSF injection may have been due to dilution of endogenous CCK released into CSF during the meal. To determine the percent recovery from CSF of exogenous CCK-8, CSF samples from CM were collected during 3 hr continuous LV injections of CCK-8 and inulin (for measurement of bulk absorption). Only 20 to 40 percent of administered CCK-8 was recovered in CM CSF. The loss of CCK-8 was probably not due to degradation in the CSF by proteolytic enzymes, since CCK-8 concentrations did not decrease during in vitro incubation at 37°C for up to 24 hr. We propose that CCK-8 is released during feeding into the ventricular system, and subsequently taken up from CSF by specialized ependymal cells for transport to sites of action.     

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.     

Opposite effects of cholecystokinin octapeptide (CCK-8) and tetrapeptide (CCK-4) after injection into the caudal part of the nucleus accumbens or into its rostral part and the cerebral ventricles

Neurons with colocalized cholecystokinin and dopamine are present predominantly in the ventral tegmental area and project mainly to the caudal part of the medial nucleus accumbens. The activity of this dopamine system can be evaluated by means of the intracranial self-stimulation behavior on male Wistar rats having chronic electrodes implanted into the medial forebrain bundle in the postero-lateral area of the hypothalamus. The direct injection of 150 pmol CCK-8 into the medio-caudal accumbens induced an increase of intracranial self stimulation while a similar administration into its rostral portion produced a slight decrease of intracranial self-stimulation. The administration of 300 pmol CCK-4 into the same medio-caudal part of the accumbens produced an inhibitory action on intracranial self stimulation lasting for 25 min. The injection of 70 to 1300 pmol CCK-4 into the cerebral ventricles produced no change on intracranial self-stimulation. The intracerebroventricular injection of 70 pmol CCK-8 induced a large decrease of intracranial self-stimulation lasting for 20 min. Sodium chloride 0.15 M or unsulphated CCK-8 injection were without effect in either case. These results support the ideas that intracerebroventricular CCK-8 injection inhibits accumbens dopaminergic activity but that CCK-8 injection into the medio-caudal part of the accumbens, where nerve terminals with colocalized CCK and DA are present, facilitates this dopaminergic activity. In addition at the level of medio-caudal accumbens, CCK-8 and CCK-4 have opposite effects.     

Specific photoaffinity crosslinking of [125I]cholecystokinin to pancreatic plasma membranes. Evidence for a disulfide-linked Mr 76 000 peptide in cholecystokinin receptors

In rat pancreatic plasma membranes, preincubated with [125I]cholecystokinin-33 (CCK-33) and washed free of unbound tracer, the irradiation by UV light induced the irreversible binding of radioactivity to high molecular weight peptides as shown by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS) and autoradiography. This was not observed when the membranes were preincubated in the simultaneous presence of [125I]CCK-33 and of either an excess of unlabelled CCK-8 or of guanosine 5'-(beta, gamma-imido)-triphosphate. The radioactivity was mostly crosslinked with a Mr 96,000 peptide and peptide species of Mr greater than 200,000, after SDS solubilization in the absence of beta-mercaptoethanol. Peptide reduction with beta-mercaptoethanol converted the high molecular weight radioactive species into a Mr 76,000 peptide that contained as much as 65% of the radioactivity crosslinked. The Mr 76,000 peptide appears, therefore, to be a disulfide-linked constituent of rat pancreatic cholecystokinin receptors.     

CCK－8和NDAP对大鼠脑和脊髓组织c－fos表达的影响

为探讨八肽胆囊收缩素（ＣＣｋ－８）和阿片肽相互作用的分子机理,利用抗体免疫沉淀技术研究了ＣＣＫ－８与ＮＤＡＰ（ｋ阿片受体激动剂）对大鼠脑（去皮层和小脑）和脊髓背柱组织Ｆｏｓ蛋白的影响。结果表明,０．１μｍｏｌ／ＬＣＣＫ－８可显著刺激脑和脊髓组织中Ｆｏｓ蛋白增加（分别是对照组的３．８倍和３．６倍）。相同浓度的ＮＤＡＰ对Ｆｏｓ蛋白的生成亦有一定的诱导作用,分别是对照组的２．７倍和２．６倍。ＣＣＫ－８和ＮＤＡＰ共同处理组织,Ｆｏｓ蛋白生成水平相似（脑）或高于（脊髓）ＣＣＫ~－８单独诱导的水平。结果表明,ＣＣＫ－８和ＮＤＡＰ均可直接诱导大鼠脑和脊髓组织ｃ－ｆｏｓ的表达,它们对ｃ－ｆｏｓ表达的相互作用在脑和脊髓中呈现不同的模式。     

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.     

Preparation of an N-acetyl-octapeptide of cholecystokinin: The role of N-acetylation in protecting the octapeptide from degradation by smooth muscle tissues

The C-terminal octapeptide of cholecystokinin (CCK-8) was acetylated on its lone N-terminal amino group using acetic anhydride in N,N-dimethylformamide. The acetylated derivative (Ac-CCK-8) and unreacted CCK-8 were separated from acetic anhydride and other reaction products by fractionation on Sephadex LH-20. Final purification was by thin-layer isoelectric focusing in a pH 2.5–4.0 gradient. The immunochemical properties of the octapeptide were unaffected by acetylation as measured by radioimmunoassay. The N-acetylated-octapeptide was equally as effective as unmodified CCK-8 in producing concentratiion-dependen isometric tension development in isolated cat gallbladder strips. Acetylation did, however, protect CCK-8 from N-terminal degradation by soluble peptidases that eluted from gallbladder and other smooth muscle tissues of the cat. Unmodified CCK-8 was degraded rapidly in the presence of these tissues and in buffers previously exposed to the same tissues. In contrast, the Ac-CCK-8 was resistant to N-terminal degradation under the same conditions. Degradation of CCK-8 from its N-terminus produces biologically inactive derivatives and could adversely affect in vitro studies. Since the acetylated-CCK-8 retained full biological and immunological activity, its use would eliminate the effect of extracellular proteolysis on CCK-8 action.     

MK-329 blocks the inhibition of alcohol intake by CCK-8

Peripheral administration of sulfated cholecystokinin octapeptide (CCK-8) potently reduces alcohol intake, preference, and blood levels in rats. MK-329 (L-364,718 or Devazepide) acts at peripheral cholecystokinin (CCK_A) receptors to antagonize CCK-8's physiological and behavioral effects, such as pancreatic stimulation and inhibition of feeding. We determined whether CCK_A receptor blockade would also prevent CCK-8's alcohol satiety effect. Water-deprived female and male rats (n = 7 for each) received randomized combinations of intraperitoneal injections of MK-329 (0, 100, 200, or 400 μg/kg) followed by CCK-8 (0 or 4 μg/kg). Rats were then given access to 5% w/v ethanol for 30 min, followed by 30-min access to water, with food ad lib. MK-329 at all doses significantly (p < 0.05) reduced the suppression of alcohol intake and food intake by CCK-8. MK-329 alone increased alcohol intake at 400 μg/kg, and increased food intake, in females and males at 100 and 200 μg/kg, respectively. We concluded that CCK-8's alcohol and food satiation effects depend on specific, peripheral CCK_A receptors, and satiation of alcohol consumption and drinking-associated feeding reflect an endogenous functional interaction of CCK-8 with CCK_A receptors.     

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.     

八肽胆囊收缩素对抗mu和Kappa型受体介导的镇痛作用

以往的资料表明,八肽胆襄收缩素(CCK-8)能对抗阿片镇痛,本实验进一步分析 CCK-8对抗哪一类型阿片受体激动剂的镇痛作用。给大鼠脊髓蛛网膜下腔(I.T.)注射 CCK-8(剂量4ng到1.0μg)既不产生痛敏也不产生镇痛。I.T.注射特异性的μ受体激动剂 PL01710 ng 或 k 受体激动剂 NDA P500 ng 引起的镇痛作用可被注射 CCK-8 4ng 所对抗。而L.T.注射δ受体激动剂 DPDPE(6.5,13.0和26.Oμg)引起的镇痛作用不能被 CCK-8(4ng,40ng I.T.)所对抗。但 CCK-8对抗 PL017和 NDAP 镇痛的作用可被 I.T.CCK 受体拮抗剂 proglumide(3μg)所翻转。以上结果表明,I.T.注射 CCK-8可有效地对抗μ和 k 受体介导的镇痛,并且这种对抗作用是经 CCK 受体介导而实现的。     

Modulation of [3H]-dopamine binding by cholecystokinin octapeptide (CCK-8)

Cholecystokinin-octapeptide (CCK-8) is a putative neurotransmitter which has been demonstrated previously to occur in midbrain dopamine neurones. We observe that CCK-8 causes changes in both the affinity and density of binding sites for [³H]-dopamine in rat striatal homogenates, in vitro, upon incubation with the peptide at a concentration of 1 micromolar. A dose-response study of the competetion of CCK-8 with [³H]-dopamine binding indicates an IC₅₀ for the peptide of 450 nM; desulfated CCK-8 and the related peptide caerulin are at least 4-fold less active than CCK-8. CCK-8 was also administered to rats in a separate study; the binding of [³H]-dopamine was evaluated to homogenates of striata and olfactory tubercles obtained from these animals, which had been treated with systemic injection at a dose of 20 micrograms/kg, daily, for four days. A decrease in the number of striatal binding sites for the radioligand was observed, with a concomitant increase in the number of binding sites in the olfactory tubercle. These data collectively suggest a possible regulatory role for CCK-8 in the ascending dopamine systems.     

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.     

N-Carboxyacyl analogues of CCK with a substituted Gly: Interaction with pancreatic and gallbladder CCK receptors

It has been reported that certain N-carboxyacyl analogues of CCK-8 and of CCK-7 with a substituted Gly in position 3 or 4 of the peptide possess higher potencies at stimulating pancreatic enzyme secretion than at stimulating gallbladder contraction, suggesting that these analogues are able to differentiate subtypes of CCK_A receptors. However, no studies examined directly the interaction of these peptides with the CCK receptors in both tissues. In the present study, CCK-8 and various N-carboxyacyl analogues of CCK-7 and of CCK-8 were prepared by solid phase synthesis using Fmoc chemistry and were purified by HPLC; molecular weight and sufficient sulfation were determined by mass spectrometry. [¹²⁵I]Bolton-Hunter(BH)-CCK-8 binding to sections of the guinea pig pancreas and gallbladder was determined under identical conditions; amylase release from pancreatic acini and contraction of gallbladder muscle strips were measured in vitro. Each peptide stimulated amylase release (EC₅₀):

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). The same relative potencies were found for stimulation of gallbladder contraction, and for the inhibition of [¹²⁵I]BH-CCK-8 binding to pancreas and gallbladder sections. These data demonstrate that the CCK_A receptors in the pancreas and on gallbladder smooth muscle possess similar affinities for the various N-carboxyacyl analogues of CCK-7 and CCK-8 with a substituted Gly and provide further evidence that the CCK_A receptors in gallbladder and pancreas cannot be distinguished pharmacologically.     

Conformational and molecular modeling studies of sulfated cholecystokinin-15

Conformational features of the C-terminal carboxyamidated pentadecapeptide of CCK (S(19)HRISDRD[SO(4)]-YMGWMDF(33)-NH(2)) were determined by NMR spectroscopy in a zwitterionic membrane-mimetic solvent system, composed of DPC micelles. The C-terminal octapeptide consisted of a well-defined pseudohelix that was nearly identical to the structure previously reported for nonsulfated CCK-8 in the same solvent system. N-terminal amino acids of CCK-15 were highly disordered, with no clear conformational preference. Extensive NOE-restrained molecular dynamics simulations of the CCK-15/CCK(1)-R complex suggested that almost all the experimentally determined intermolecular contact points provided by NMR, site-directed mutagenesis, and photoaffinity labeling could be simultaneously satisfied, when the N-terminus of the ligand is placed in close spatial proximity to the N-terminus of the receptor.