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.     

八肽胆囊收缩素对吗啡抑制大鼠离体空肠电与收缩活动的对抗作用

本研究探讨了八肽胆囊收缩素（ＣＣＫ－８）对抗吗啡对大鼠离体空肠电与收缩活动的作用。结果表明,吗啡能抑制ＡＣｈ对空肠峰波发放和收缩的加强作用;ＣＣＫ－８可对抗吗啡的作用;在此基础上,ＣＣＫ－Ａ受体拮抗剂ｄｅｖａｚｅｐｉｄｅ（１０ｎｍｏｌ／Ｌ）能完全翻转ＣＣＫ－８的抗吗啡作用,但是ＣＣＫ－Ｂ受体拮抗剂Ｌ－３６５,２６０在１０ｎｍｏｌ／Ｌ时可部分翻转、在３０ｎｍｏｌ／Ｌ时能完全翻转ＣＣＫ－８的作用。上述     

八肽胆囊收缩素(CCK_8)的人工合成

本文报道了用液相法合成八肽胆囊收缩素(H-Asp-Tyr(SO_3H)-Met-Gly-Trp-Met-Asp-Phe-NH_2,ccK_8)。先分别合成C-端四肽和N-端四肽,然后缩合成八肽,并用温和的乙酰硫酸吡啶盐(PAS)法使其酪氨酸的酚羟基硫酯化。根据其氨基酸组成,层析行为和生物活性等证实产物为纯品。     

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.     

The molecular nature of vascularly released cholecystokinin from the isolated perfused porcine duodenum

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 affinitychromatography 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.     

High-relaxivity supramolecular aggregates containing peptides and Gd complexes as contrast agents in MRI

Mixed supramolecular aggregates, obtained by assembling together two amphiphilic monomers (C₁₈H₃₇)₂NCO(CH₂)₂CO(AdOO)₅-G-CCK8 (AdOO is 8-amino-3,6-dioxaoctanoic acid, CCK8 is C-terminal octapeptide of cholecystokinin) and (C₁₈H₃₇)₂NCO(CH₂)₂COLys(DTPAGlu)CONH₂ (DTPAGlu is N,N-bis[2-[bis(carboxyethyl)amino]ethyl]-l-glutamic acid), are characterized for their structural parameters by dynamic light scattering and for their relaxometric properties, in the absence and in the presence of 0.9 wt% NaCl. Two different aggregates (micelles and bilayer structures) are present in the absence of NaCl, while only bilayer structures are observed at physiological ionic strength. The presence of NaCl increases the ionic strength, promoting a decrease in the repulsions between the polar heads and among the aggregates in solution, thus supporting the formation of large-curvature aggregates such as bilayer structures like vesicles. In these conditions the closed, vesicular shape and the large size (hydrodynamic radius of about 300 Å) of the aggregates allow a high number of paramagnetic gadolinium complexes and bioactive peptides to be accommodated on the inner and external surfaces . The presence of the salt causes a variation in the structural arrangement of the molecules and a partial rigidification of the assembled Gd(III) complexes on the surface vesicles, reducing their internal motions and giving an approximately 15% higher relaxivity value (r _1p = 21.0 and 18.6 Mm^?1 s^?1 in the presence and in the absence of NaCl, respectively). The vesicles obtained, for the high relaxivity of each gadolidium complex and for the presence of a surface-exposed bioactive peptide, are very promising candidates as target-selective MRI contrast agents.     

Ceruletide, ceruletide analogues and cholecystokinin octapeptide (CCK-8): effects on isolated intestinal preparations and gallbladders of guinea pigs and mice

The smooth muscle stimulatory effects of cholecystokinin octapeptide (CCK-8), ceruletide (CER), ten analogues of CER, and carbachol were studied in isolated organs of the guinea pig and the mouse (stomach, ileum, duodenum, colon and gallbladder). On a molar basis, CCK-8 and CER had in all organs except stomach greater potency (lower EC50) than carbachol. The effectiveness (Emax) of CCK-8 and CER was in the gut less than that of carbachol, in the guinea pig gallbladder equal with and in the mouse gallbladder superior to that of carbachol. The alteration of peptide structure was virtually without influence on effectiveness; however, it greatly modified the potency and the organ selectivity of the effect. There was no clear-cut correlation between the potency to stimulate smooth muscle and to alter the behavior of the mouse.     

Protective effect of cholecystokinin octapeptide on angiotensin II-induced apoptosis in H9c2 cardiomyoblast cells

Cholecystokinin (CCK) and its receptors are expressed in mammalian cardiomyocytes and are involved in cardiovascular system regulation; however, the exact effect and underlying mechanism of CCK in cardiomyocyte apoptosis remain to be elucidated. We examined whether sulfated CCK octapeptide (CCK-8) protects H9c2 cardiomyoblast cells against angiotensin II (Ang II)-induced apoptosis. The H9c2 cardiomyoblasts were subjected to Ang II with or without CCK-8 and the viability and apoptotic rate were detected using a Cell Counting Kit-8 assay, Hoechst 33342 staining, terminal deoxyribonucleotide transferase-mediated nick-end labeling assays, and flow cytometry. In addition, specific antiapoptotic mechanisms of CCK-8 were investigated using specific CCK1 (Devazepide) or CCK2 (L365260) receptor antagonists, or the PI3K inhibitor LY294002. The expression of CCK, CCK1 receptor, CCK2 receptor, Akt, p-Akt, Bad, p-Bad, Bax, Bcl-2, and caspase-3 were detected by Western blot analysis and real-time polymerase chain reaction. We found that CCK and its receptor messenger RNA (mRNA) and protein are expressed in H9c2 cardiomyoblasts. Ang II-induced increased levels of CCK mRNA and protein expression and decreased levels of CCK1 receptor protein and mRNA. Pretreatment of CCK-8 attenuated Ang II-induced cell toxicity and apoptosis. In addition, pretreatment of H9c2 cells with CCK-8 markedly induced expression of p-Akt, p-bad, and Bcl-2 and decreased the expression levels of Bax and caspase-3. The protective effects of CCK-8 were partly abolished by Devazepide or LY294002. Our results suggest that CCK-8 protects H9c2 cardiomyoblasts from Ang II-induced apoptosis partly via activation of the CCK1 receptor and the phosphatidyqinositol-3 kinase/protein kinase B (PI3K/Akt) signaling pathway.     

In vitro degradation of the C-terminal octapeptide of cholecystokinin by 'enkephalinase A'

As the C-terminal octapeptide of cholecystokinin represents a putative neurotransmitter in the central nervous system, the membrane-bound enzymes involved in its inactivation were investigated. Two aminopeptidases, involved in the cleavage of enkephalins, and a metalloendopeptidase were identified in extracts of solubilized synaptic membranes. The metalloendopeptidase, which cleaves CCK-8 at the Trp30-Met31 bond, appeared to be indistinguishable from 'enkephalinase A1' on the basis of its chromatographic behaviour, sensitivity to inhibitors and relative affinities for Met- and Leu-enkephalins. This finding indicates that CCK-8 is inactivated in vitro by the same peptidases as enkephalins.     

Multiple treatment potentiates the anticonvulsive activity of cholecystokinin octapeptides

The dose-response curves for the anticonvulsive activity of sulfated and nonsulfated cholecystokinin octapeptide (CCK-8-SE and CCK-8-NS) against picrotoxin-induced (6 mg/kg SC) seizures were assessed either following or without pretreatment with a single high dose of CCK-8-SE or CCK-8-NS, to examine acute tolerance to the effect after IP injections in mice. As CCK-8-SE or CCK-8-NS pretreatment, a 1.6 μmole/kg dose was injected 2 hr prior to the second injection. No acute tolerance to the anticonvulsive activity was demonstrated, and CCK-8-NS pretreatment significantly potentiated its own anticonvulsive activity. Chronic (8-day) daily treatment with a 0.16 μmole/kg dose of CCK-8-SE or CCK-8-NS antagonized seizures by picrotoxin, presumably in a cumulative manner. To investigate the interactions of CCK octapeptides with other anticonvulsive agents, picrotoxin-induced seizures were antagonized with several doses of diazepam following or without acute, high-dose pretreatment with CCK-8-SE or CCK-8-NS. The two octapeptides only slightly modified the activity of diazepam: CCK-8-SE pretreatment displayed a tendency to antagonize it, while CCK-8-NS pretreatment to potentiate it. The results suggest that multiple treatment with CCK-8 induces sensitization of CCK receptors mediating anticonvulsive activity.     

八肽胆囊收缩素对大鼠颈动脉窦压力感受器反射的抑制作用

在36只隔离灌流颈动脉窦区的麻醉大鼠上,观察了八肽胆囊收缩素(cholecystokinin octapepide,CCK-8)对颈动脉窦压力感受器反射的影响。其结果如下：(1)以CCK-8(0．1、0．5、1．0μmol／L)隔离灌流颈动脉窦区时,压力感受器机能曲线向右上方移位,曲线最大斜率(peak slope,PS)减小,反射性血压下降幅度(reflex decrease,RD)减少,阈压(threshold pressure,TP)和饱和压(saturation pressure,SP)均增高。其中RD、PS和TP呈明显的剂量依赖性;(2)用CCK-8的非特异性受体拮抗剂丙谷胺(100μmol／L)预处理后,能明显减弱CCK-8(0．50mol/L)对压力感受器反射的抑制;(3)预先灌流一氧化氮合酶(nitric oxide synthase,NOS)阻断剂(L-NAME,100μmol／L),不能阻断CCK-8(0．5μmol/L)对压力感受器反射的影响;(4)用Ca^2 通道激动剂Bay K 8644(500nmol／L)预处理后,也能明显减弱CCK-8(0．5μmol／L)对压力感受器反射的抑制作用。以上结果提示,CCK-8是通过作用于颈动脉窦压力感受器神经元末梢上的受体而起到抑制作用的,其机制可能为抑制了牵张敏感性通道,致使Ca^2 离子内流减少,而与内皮细胞释放NO无关。     

Basic Amino Acids at the C-Terminus of the Third Intracellular Loop Are Required for the Activation of Phospholipase C by Cholecystokinin-B Receptors

Abstract: In common with other G_q protein-coupled receptors, the third intracellular loop of the cholecystokinin-B (CCK-B) receptor contains three basic amino acids (K333/K334/R335) at the C-terminal segment. To determine the importance of these conserved basic residues in G_q-protein activation and stimulation of phospholipase C, these basic amino acids were mutated. Subsequently, the ability of resulting mutant receptors to activate phospholipase C was investigated by measuring inositol phosphate formation in COS-7 cells and recording Ca²⁺-activated Cl^? currents from Xenopus oocytes. Site-directed mutagenesis was performed to mutate the three basic amino acids, K333/K334/R335, to neutral amino acids, M333/T334/L335. When the resulting mutant CCK-B receptors were expressed in COS-7 cells and Xenopus oocytes, sulfated cholecystokinin octapeptide (CCK-8) failed to induce inositol phosphate formation in COS-7 cells and evoke Ca²⁺-activated Cl^? currents from oocytes. Each basic amino acid was also mutated (K333M, K334T, and R335L). All three single-point mutations resulted in a significant reduction in CCK-8-induced inositol phosphate formation and CCK-8-activated Ca²⁺-dependent Cl^? currents. It is interesting that substituting the basic amino acids, K333/K334/R335, with three other basic residues, R333/R334/K335, did not change the maximal CCK-8-simulated inositol phosphate formation and the amplitude of CCK-8-evoked Ca²⁺-dependent Cl^? currents. Radioligand-binding studies showed that the above-mentioned mutations did not affect the affinity for CCK-8 and receptor expression level in COS-7 cells. These findings suggest that basic amino acids at the C-terminus of the third cytoplasmic loop are required for the signal transduction by CCK-B receptors.     

兔侧脑室注射八肽胆囊收缩素抗血清对血浆自由脂肪酸浓度的影响

本工作根据抗原抗体间具有高度特异性的中和作用的原理,将微量 CCK-8抗血清注射入制备有埋藏套管的慢性实验兔的侧脑室内,观察在中和脑内外源性或内源性的 CCK-8后,血浆 FFA 浓度的变化,结果如下:1.侧脑室内注射正常兔血清,对血浆 FFA 浓度无明显影响;在注射 CCK-8同时注射兔正常血清,也不影响 CCK-8降低血浆 FFA 浓度的作用。2.侧脑室内注射 CCK-8抗血清能有效地阻断外源性脑室注射 CCK-8降低血浆 FFA 的作用,此阻断作用随 CCK-8抗血清剂量的增加而增强。3.侧脑室内单独注射 CCK-8抗血清,血浆 FFA 浓度明显升高,这一作用可能是由于中和了脑内内源性 CCK-8的作用所致。以上结果表明,CCK-8脑室注射引起的血浆 FFA 降低的作用具有一定的特异性;而在正常生理情况下,脑内释放的内源性 CCK-8可能参与血浆 FFA 代谢的调节。     

Effect of acetylation on antioxidant and cytoprotective activity of polysaccharides isolated from pumpkin (Cucurbita pepo, lady godiva)

Acetylation of pumpkin (Cucurbita pepo, lady godiva variety) polysaccharide using acetic anhydride with pyridines as catalyst under different conditions was conducted to obtain different degrees of acetylation on a laboratory scale. Furthermore, antioxidant activities and cytoprotective effects of pumpkin polysaccharide and its acetylated derivatives were investigated employing various established in vitro systems. Results showed that addition of pyridine as catalyst could increase the degree of substitution, whereas volume of acetic anhydride had little effect. The acetylated polysaccharides in DPPH scavenging radical activity assay, superoxide anion radical activity assay and reducing power assay exhibited higher antioxidant activity than that of unmodified polysaccharide. H₂O₂-induced oxidative damages on rat thymic lymphocyte were also prevented by pumpkin polysaccharide and its acetylated derivatives and the derivatives presented higher protective effects. On the whole, acetylated polysaccharide showed relevant antioxidant activity both in vitro and in a cell system.     

The effects of γ-hexachlorocyclohexane on amylase secretion and inositol phospholipid metabolism in mouse pancreatic acini

Dispersed mouse and guinea-pig pancreatic acini were used to examine the effects of the inositol analogue, γ-hexachlorocyclohexane (lindane) on agonist-stimulated amylase secretion. Secretion from mouse acini in response to carbachol and cholecystokinin octapeptide (CCK-8) was reduced by lindane. Similarly, amylase release from guinea-pig acini stimulated by carbachol was abolished by lindane. These acini, however, still remained responsive to dibutyryl-cAMP with only a slightly diminished secretion to this agent. Inositol phospholipid synthesis and hydrolysis was stimulated in mouse acini by both carbachol and CCK-8. Although hydrolysis of these lipids in response to CCK-8 was reduced by only 18%, stimulation of inositol phospholipid synthesis by either agonist was abolished by lindane. Dose-response curves for inositol phospholipid synthesis stimulated by carbachol and CCK-8 in mouse acini were biphasic and superimposable with those of amylase secretion. In contrast, the dose-response curve for phosphoinositide hydrolysis was sigmoid and clearly separable from that of synthesis. Reducing the external Ca²⁺ concentration caused the dose-response curves for carbachol- and CCK-8-induced inositol phospholipid synthesis to be displaced to the right, as has been observed for amylase secretion. A23187 was also found to induce amylase secretion and inositol phospholipid synthesis, and both of these responses were inhibited by lindane. Amylase secretion and inositol phospholipid synthesis may, therefore, be closely related events in the exocrine pancreas. Lindane may provide a valuable tool with which to determine the role of inositol phospholipid metabolism in stimulus-response coupling.     

CCK-8对内毒素休克大鼠肺脏细胞因子的抑制效应

观察八肽胆囊收缩素(cholecystokinin-octapeptide,CCK-8）改善脂多糖(lipopolysaccharide,LPS）引起的大鼠内毒素性休克（endotoxic shock,ES）过程中血清及肺脏细胞因子的变化,探讨p38比裂素活化蛋白激酶（p38 mito-gen-activated protein kinase,p38 MAPK）的信号转导作用。用生理多道记录仪观察尾静脉注入LPS(p38 mito-gen-activated protein kinase,p38 MAPK）的信号转导作用。用生理多道记录仪观察尾静脉注入 LPS（8mg/kg i.v.）复制的SD大鼠ES模型、LPS注入前10min尾静脉注入CCK-8(40ug/kg i.v.）、单独注入CCK-8(40Uug/kg i.v.）或生理盐水（对照）的四组大鼠平均动脉血压（MAP）的改变,应用ELISA试剂盒检测血清和肺脏中炎性细胞因子（TNF-a、IL-1β和IL-6）的变化。用Western blot检测肺脏p38 MAPK的表达。结果显示：CCK-8可改善LPS引起的大鼠MAP的下降。与对照组相比,LPS可显著增加血清和肺脏TNF-a、IL-1β和IL-6含量;CCK-8可显著抑制LPS诱导的血清和肺脏TNF-a、IL-1β和IL-6的增加。CCK-8可增加ES大鼠肺脏磷酸化p38 MAPK的表达。结果提示CCK-8可改善ES大鼠MAP的降低,并对肺脏促炎性细胞因子过量产生有抑制作用,p38MAPK可能参与了其信号转导机制。     

Blocking of cholecystokinin octapeptide behavioral effects by proglumide

An open field apparatus was used to assess the effect of proglumide, a selective antagonist of cholecystokinin octapeptide (CCK-8), to block the behavioral effect of CCK-8 in rats. Intracerebroventricular (ICV) injection of CCK-8 (0.5 to 2 micrograms) was effective in suppressing general exploratory activities and this effect was blocked by proglumide at doses of 2 to 5 micrograms administered ICV or 1 mg/kg administered subcutaneously. The effect of peripherally administered CCK-8 (10 micrograms/kg) was blocked by peripherally administered proglumide at a dose of 2 mg/kg but not by centrally administered proglumide at a dose of 5 micrograms/rat. The behavioral effect of CCK-8 was thus clearly blocked by proglumide.     

Heterocycles from saccharide hydrazones : Part I. Saccharide 1,3,4-oxadiazoles

Oxidation, with iodine-mercuric oxide, of acetylated saccharide aroylhydrazones and of aromatic aldehyde hydrazones yields 5-aryl-2-(polyacetoxyalkyl)-1,3,4-oxadiazoles and 2,5-diaryl-1,3,4-oxadiazoles, respectively. On de-O-acetylation, saccharide oxadiazole acetates rearrange to the tautomeric, cyclic iminolactones which, on reacetylation, regenerate the starting oxadiazoles. Attempted dehydration of saccharide acetyl- and benzoyl-hydrazones by treatment with boiling acetic anhydride under reflux resulted in the formation of peracetylated N,N-diacetylhydrazones and-N-acetyl-N-benzoylhydrazones, respectively.     

Regional distributions of somatostatin and cholecystokinin-like immunoreactivities in rat and bovine brain

The distribution of somatostatin- and cholecystokinin octapeptide (CCK-8)-like immunoreactivities in the central nervous system of bovine and rat has been studied using a sensitive and specific radioimmunoassay. The most interesting information is the high concentration of CCK-8 in the various cortical areas in rat and bovine. The nuclei of amygdala contain the highest amount of octapeptide in rat while in bovine brain, this structure contains the second highest concentration after polus frontalis.