胆囊收缩素8对大鼠大脑皮质细胞钙调素和蛋白激酶C活性的影响

 为阐明中枢神经系统中胆囊收缩素 8(CCK8)受体的信号传递机制 ,以分离的大鼠大脑皮质神经细胞为材料 ,观察了CCK8对细胞内钙调素 (CaM)、3′ ,5′ 环腺苷酸 (cAMP)、蛋白激酶C(PKC)活性的影响 研究结果表明 ,CCK8可刺激大脑皮质细胞CaM、PKC活性的增加 ,并有剂量依赖关系 但CCK8在 10 -12 ～ 10 -6mol L范围内 ,细胞内cAMP含量无显著变化 利用受体亚型L 364,718和L 365,2 60的研究表明 ,两种拮抗剂均可抑制CCK8引起的CaM和PKC活性变化 ,但两者IC50 不同 对于CaM ,CCKB 受体拮抗剂L 365,2 60的IC50 比CCKA 受体拮抗剂L 364,718低 4 0倍 ;而对于PKC ,L 365,2 60的IC50 比L 364,718低 60倍 因此认为 ,CCK8主要是通过CCKB 受体介导了CaM和PKC活性的变化     

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     

八肽胆囊收缩素对大鼠心功能的影响及受体机制

为探讨八肽胆囊收缩素 (CCK 8)对麻醉大鼠心功能的影响及受体机制 ,实验监测了左心室收缩压(LVP)、左心室收缩与舒张期内压变化的最大速率 (±LVdp/dtmax)、心率 (HR)和平均动脉压 (MAP)。结果如下 :小剂量CCK 8(0 4 μg/kg)可引起心动过速 ,MAP、LVP和±LVdp/dtmax轻度上升 ;中剂量CCK 8(4 μg/kg)和大剂量CCK 8(4 0 μg/kg)可引起心动过缓 ,MAP、LVP和±LVdp/dtmax显著增加 ;应用CCK 受体 (CCK R)拮抗剂丙谷胺 (1 0mg/kg)抑制以上变化 ;由逆转录 聚合酶链反应 (RT PCR)检测到心肌组织有CCK A受体 (CCK AR)和CCK B受体 (CCK BR)mRNA表达。以上结果提示 :CCK 8可激活心肌组织的CCK R ,引起剂量依赖性的心功能增加和心率改变。     

八肽胆囊收缩素的结构与活性关系

本文采用液相多肽合成法制备了八肽胆囊收缩素(CCK_8)的六种类似物,并测定了它们诱导离体的豚鼠胆囊收缩的生物学活性。发现CCK_8的N-端乙酰化不改变其生物活性,脱去N-端氨基的CCK_8类似物即Suc-CCK_7与母体CCK_8相比,其活性明显增加。在Boc-CCK_7中,Met被NIe取代活性可完全保留,Gly~(29)被D-Ala取代后仍保留相当的活性,但Gly~(29)被β-Ala取代后则失去胆囊收缩活性;在Met~(28)-Gly~(29)区域引入刚性的r-内酰胺环作为构象限制,也导致活性完全丧失。     

八肽胆囊收缩素对吗啡成瘾大鼠中枢痛觉的调制

目的:观察八肽胆囊收缩素(CCK-8)对正常及吗啡成瘾大鼠尾核中痛兴奋神经元(PEN)电活动的影响,从而进一步探讨中枢CCK-8和尾核在吗啡成瘾大鼠痛觉调制中的作用.方法:70只Wistar大鼠随机分为2组:正常对照组35只(又分为生理盐水组巧只和CCK-8组20只)及吗啡成瘾组35只、(又分为生理盐水组15只和CCK-8组20只).大鼠背部皮下注射递增剂量吗啡,依次为5、10、20、40、50、60mg/kg,3次/d(8:00,12:00,16:00),连续给药6d,建立吗啡成瘾大鼠的模型.正常对照组大鼠背部皮下注射生理盐水,时间、剂量均与吗啡成瘾组相同.第7天8:00观察大鼠的自然戒断症状30min后开始实验.实验以电脉冲刺激大鼠的坐骨神经作为伤害性痛刺激,用玻璃微电极记录尾核中PEN的放电,观察尾核内注入CCK-8对PEN电活动的影响.结果:实验结果表明:①CCK-8可提高正常大鼠尾核中PEN的兴奋性,即25个PEN平均秒净增值由注射CCK-8前(100%)增加到(224.34±10.81)5,潜伏期缩短到(54.69±5.62)%.②CCK-8使吗啡成瘾大鼠尾核中PEN的兴奋性也增高,22个PEN的平均秒净增值比注药前(100%)提高了(118.93±8.50)%,潜伏期缩短了(33.96±7.23)%.结论:CCK-8可使吗啡成瘾与正常大鼠尾核中PEN对电刺激的兴奋性增强,均呈易化疼痛作用,证明中枢CCK-8系统和尾核在吗啡成瘾过程和疼痛的调节中都起到了一定的作用.     

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。     

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。     

Localization of cholecystokinin receptor subtypes in the endocine pancreas.

This study was undertaken to clarify the controversy in the literature about pancreatic localization of the cholecystokinin (CCK) CCK(A) and CCK(B) receptors. With antibodies used by other investigators, we first established their specificity by Western blotting, indirect immunofluorescence, and confocal microscopy with each antibody's peptide antigen. Co-localization assays between the CCK receptors and the pancreatic hormones insulin, glucagon, and somatostatin revealed that the CCK(A) RAbs 1122 and R1-2 recognized insulin and glucagon cells in rat, pig, and human pancreas but not in the somatostatin cells. Conversely, the three CCK(B) RAbs tested, 9262, 9491, and GR4, identified the somatostatin cells. Abs 9491 and GR4 occasionally co-localized with glucagon, a feature that never occurred with Ab 9262. Finally, the specificity of Ab 9262 for the pancreatic CCK(B) R was confirmed in six different species. It co-localized with somatostatin but never with glucagon in these species. Our data suggest the use of Abs 1122 and 9262 to specifically identify and localize pancreatic CCK(A) and CCK(B) receptors, respectively. Confusion in the literature may result from the lack of specificity of most antibodies used, as established in this study.     

Mg2+/Ca2+-ATPase Activity Is Not Enriched in Synaptic Vesicles Isolated from Rat Cerebral Cortex

Neuronal ATPases comprise a wide variety of enzymes which are not uniformly distributed in different membrane preparations. Since purified vesicle fractions have Mg²⁺/Ca²⁺-ATPase, the purpose of the present study was to know whether such enzyme activities have a preferential concentration in a synaptic vesicle fraction in order to be used as markers for these organelles. Resorting to a procedure developed in this Institute, we fractionated the rat cerebral cortex by differential centrifugation following osmotic shock of a crude mitochondrial fraction and separated a purified synaptic vesicle fraction over discontinuous sucrose gradients. Mg²⁺/Ca²⁺-ATPase activities and ultrastructural studies of isolated fractions were carried out. It was observed that similar specific activities for Mg²⁺/Ca²⁺-ATPases were found in all fractions studied which contain synaptic vesicles and/or membranes. Although the present results confirm the presence of Mg²⁺ and Ca²⁺-ATPase activities in synaptic vesicles preparations, they do not favor the contention that Mg²⁺/Ca²⁺-ATPase is a good marker for synaptic vesicles.     

PtdIns(4,5)P2 interacts with CaM binding domains on TRPM3 N-terminus

TRPM3 has been reported to play an important role in Ca²⁺ homeostasis, but its gating mechanisms and regulation via Ca²⁺ are unknown. Ca²⁺ binding proteins such as calmodulin (CaM) could be probable modulators of this ion channel. We have shown that this protein binds to two independent domains, A35-K124 and H291-G382 on the TRPM3 N-terminus, which contain conserved hydrophobic as well as positively charged residues in specific positions, and that these residues have a crucial impact on its binding. We also showed that another Ca²⁺ binding protein, S100A1, is able to bind to these regions and that CaM and S100A1 compete for these binding sites on the TRPM3 N-terminus. Moreover, our results suggest that another very important TRP channel activity modulator, PtdIns(4,5)P₂, interacts with the CaM/S100A1 binding sites on the TRPM3 N-terminus with high affinity.     

Effects of Amino Acids and Glutathione on Rat Liver Histidase Activity in vitro

The activity of histidase (l-histidine ammonia-lyase EC 4. 3. 1. 3) of rat liver was inhibited by cystine competitively with the substrate histidine. Histidase was inactivated by preincubation with cystine, but the inactivated enzyme was reactivated by the addition of glutathione to the reaction mixture. Cysteine, at a lower level, partly reactivated the enzyme inactivated by cystine, but, at a higher level, did not reactivate it. Cysteine itself acted inhibitory on the enzyme. Glutathione did not reactivate histidase activity inhibited by cysteine. No other amino acid affected histidase activity at the level of 2 mm. Apparent Km of rat liver histidase was 1.8 × l0^?m.

Neither inhibitory nor activating effect on histidase activity was observed by the addition of liver homogenate obtained from rats fed a histidine imbalanced diet to that from those fed a basal diet and vice versa.     

Cholecystokinin-8 activates myenteric neurons in 21- and 35-day old but not 4- and 14-day old rats

Cholecystokinin (CCK) activates the myenteric neurons of adult rats. The goal of this work is to determine the ontogeny of this activation by CCK-8 in the myenteric plexus of the duodenum (2 cm immediately following the pyloric sphincter aborally) and compare it with that of the dorsal vagal complex (DVC) - which occurs in 1-day old pups. Despite the existence of both of the CCK receptors, CCK₁ and CCK₂, in 4, 14, 21 and 35 day old rats, CCK-8 (0, 5, 10, 20 and 40 μg/kg, i.p.) increased Fos-like immunoreactivity (Fos-LI, a marker for neuronal activation) in the myenteric neurons of 21- and 35-day old rats but in the DVC of all age groups. As such, this belated activation of myenteric neurons by CCK-8 compared to the DVC may reflect a delayed role for these neurons in CCK-related functions.     

Effects of Lead on Adenylate Cyclase Activity in Rat Cerebral Cortex

Lead decreased in a dose dependent manner the basal AC activity in membranes of rat cerebral cortex (IC₅₀ = 2.5 ± 0.1 M). In membranes preincubated under basal conditions, AC activity was stimulated by approximately two and fourfold by 10 M Gpp(NH)p or forskolin, respectively. Under basal conditions, lead (3 M) inhibited enzyme activity up to 50%, but was not able to inhibit the Gpp(NH)p- or the forskolin-stimulated AC activity. However, in membranes preincubated with Gpp(NH)p (10 M), lead (3 M) had no significant effect on enzyme activity, but it partly blocked the stimulation of AC activity elicited by forskolin (10 M). In membranes preincubated with 10 M lead, the addition of 10 M Gpp(NH)p or forskolin in the incubation medium did not stimulate AC activity. However, when added together in the incubation medium Gpp(NH)p + forskolin produced an increase in enzyme activity. In membranes preincubated with 10 M lead + 10 M Gpp(NH)p, Gpp(NH)p (10 M) or forskolin (10 M) added alone or in combination to the incubation medium did not stimulate AC activity. Moreover, under these latter conditions lead had no further effect on enzyme activity. These results indicate that lead may interact with G-proteins and with the catalytic subunit of cerebral cortical AC to produce inhibition of the enzyme activity.     

An ACTH-Like Peptide Immunocortin: Effect on the Activity of Cells from the Rat Adrenal Cortex

The effect of immunocortin, an ACTH-like decapeptide VKKPGSSVKV corresponding to the 11–20 sequence of the variable part of the human IgG1 heavy chain on the content of 11-hydroxycorticosteroids (CS) in rat adrenal glands and blood serum in vivo was studied. An intramuscular injection of immunocortin at a dose of 10 g/kg was found in an hour to induce a twofold decrease in CS content in the adrenal glands and a 1.8-fold increase in the blood serum CS content. At the same time, an immunocortin dose of 100 g/kg exerted practically no effect on the CS content and its dose of 1000 g/kg increased the CS content both in adrenal glands and in blood serum by 1.6 and 2.2 times, respectively. Four hours after the injection of any of the three doses of immunocortin, the CS content in adrenal glands did not differ from the control value, and after 24 h the content decreased threefold. Immunocortin was shown to be bound by the ACTH receptors in the membranes of the rat adrenal cortex with a high affinity and specificity (inhibiting the specific binding of ¹²⁵I-labeled ACTH-(11–24) peptide with K _i of 1.2 nM).     

Evidence That Antioxidants Prevent the Inhibition of Na+,K+-ATPase Activity Induced by Octanoic Acid in Rat Cerebral Cortex in Vitro

The objective of the present study was to investigate the in vitro effects of octanoic acid, which accumulates in medium-chain acyl-CoA dehydrogenase (MCAD) deficiency and in Reye syndrome, on key enzyme activities of energy metabolism in the cerebral cortex of young rats. The activities of the respiratory chain complexes I–IV, creatine kinase, and Na⁺, K⁺-ATPase were evaluated. Octanoic acid did not alter the electron transport chain and creatine kinase activities, but, in contrast, significantly inhibited Na⁺, K⁺-ATPase activity both in synaptic plasma membranes and in homogenates prepared from cerebral cortex. Furthermore, decanoic acid, which is also increased in MCAD deficiency, and oleic acid strongly reduced Na⁺, K⁺-ATPase activity, whereas palmitic acid had no effect. We also examined the effects of incubating glutathione and trolox (-tocopherol) alone or with octanoic acid on Na⁺, K⁺-ATPase activity. Tested compounds did not affect Na⁺, K⁺-ATPase activity by itself, but prevented the inhibitory effect of octanoic acid. These results suggest that inhibition of Na⁺, K⁺-ATPase activity by octanoic acid is possibly mediated by oxidation of essential groups of the enzyme. Considering that Na⁺, K⁺-ATPase is critical for normal brain function, it is feasible that the significant inhibition of this enzyme activity by octanoate and also by decanoate may be related to the neurological dysfunction found in patients affected by MCAD deficiency and Reye syndrome.