粉防己碱和蝙蝠葛碱对人白血病细胞株HL－60和K_（562）的生长抑制作用

研究粉防己碱（Ｔｅｔ）和蝙蝠葛碱（Ｄａｎ）对人早幼粒细胞白血病ＨＬ－６０和人红白细胞白血病Ｋ５６２增长的影响，并与维拉帕米（Ｖｅｒ）和三氟拉嗪（ＴＦＰ）比较。结果Ｔｅｔ和Ｄａｕ对ＨＬ－６０和Ｋ５６２的增长有很强抑制作用，呈浓度依赖性，ＩＣ５０分别为３．０，４．４ｍｇ·Ｌ－１和３．３，５．６ｍｇ·Ｌ－１；Ｔｅｔ还抑制ＨＬ－６０细胞的分裂相。同样条件下Ｖｅｒ和ＴＦＰ对ＨＬ－６０和Ｋ５６２也有抑制作用，但较前两者为弱。Ｔｅｔ和Ｄａｎ为双苄基异喹啉生物碱，已被阐明有Ｃａ２＋拮抗剂和ＣａＭ抑制剂活性，提示其抑制肿瘤细胞生长与此有关，并有可能代替常用的Ｃａ２＋拮抗剂Ｖｅｒ与化疗药联合应用于肿瘤的治疗。     

粉防己碱和蝙蝠葛碱对人白血病细胞株HL－60和K_（562）的生长抑制作用

研究粉防己碱（Ｔｅｔ）和蝙蝠葛碱（Ｄａｎ）对人早幼粒细胞白血病ＨＬ－６０和人红白细胞白血病Ｋ５６２增长的影响，并与维拉帕米（Ｖｅｒ）和三氟拉嗪（ＴＦＰ）比较。结果Ｔｅｔ和Ｄａｕ对ＨＬ－６０和Ｋ５６２的增长有很强抑制作用，呈浓度依赖性，ＩＣ５０分别为３．０，４．４ｍｇ·Ｌ－１和３．３，５．６ｍｇ·Ｌ－１；Ｔｅｔ还抑制ＨＬ－６０细胞的分裂相。同样条件下Ｖｅｒ和ＴＦＰ对ＨＬ－６０和Ｋ５６２也有抑制作用，但较前两者为弱。Ｔｅｔ和Ｄａｎ为双苄基异喹啉生物碱，已被阐明有Ｃａ２＋拮抗剂和ＣａＭ抑制剂活性，提示其抑制肿瘤细胞生长与此有关，并有可能代替常用的Ｃａ２＋拮抗剂Ｖｅｒ与化疗药联合应用于肿瘤的治疗。     

异喹啉类化合物的合成、生物活性与构效关系研究

结合我室异喹啉类化合物心血管活性的研究，主要就双苄基异喹啉类、原小蘖碱共和单苄基异喹啉。类及有关化合物的钙拮抗、③上腺素能ａ、β受体调控、抗心律失常、降压和抗血小板聚集等心血管作用进行了综述。阐述了某些化合物的作用机理和构效关系，还介绍了异喹啉类化合物的三种制备法，     

和厚朴酚对钙调素拮抗作用的研究

采用钙调素(CaM)依赖性环核苷酸磷酸二酯酶及丹磺酰标记CaM,研究和厚朴酚对CaM的作用.发现和厚朴酚能抑制CaM刺激环核苷酸磷酸二酯酶的活性,随着CaM浓度的增加,IC_(50)也相应增加。在Ca~(2+)存在下.和厚朴酚能降低丹磺酰标记的CaM的荧光强度,使其荧光发射光谱峰位红移。实验结果提示,和厚朴酚在Ca~(2+)存在下,能与CaM结合.从而拮抗其对靶酶——磷酸二酯酶的激活。另外,和厚朴酚对CaM依赖性磷酸二酯酶基础活性具有刺激作用。     

马兜铃酸与钙调素相互作用的荧光光谱分析

自从Cheung发现钙调素(Calmodulin,CaM)以来,钙调素拮抗剂在研究钙调素分子调节机制方面发挥了重要作用。马兜铃酸是我室新近发现的一种钙调素拮抗剂,它是马兜铃科植物马兜铃(Aristolochia debilisSieb. et Zucc.)中分离提纯得到的单体化合物,具有抗肿瘤和提高细胞免疫的作用。我室利用马兜铃酸(Aristolochic acid)和丹磺酰标记的钙调素(D-CaM)相互作用产生的荧光光谱变化进行了一系列的研究,     

阿片δ受体选择性不可逆拮抗剂FIT和SuperFIT的新法合成

报道了阿片δ受体选择性不可逆拮抗剂ＦＩＴ（１）,ＳｕｐｅｒＦＩＴ（２）和ｃｉｓ-（±）-ＳｕｐｅｒＦＩＴ（３）的合成,并提出了一种通过酰仲胺与ＣＳ２在ＮａＯＨ／Ｋ２ＣＯ３／ＭｅＣＮ体系中反应制备异硫氰基芬太尼类似物的合成新方法。     

异喹啉类化合物的合成、生物活性与构效关系研究

结合我室异喹啉类化合物心血管活性的研究,主要就双苄基异喹啉类、原小蘖碱共和单苄基异喹啉。类及有关化合物的钙拮抗、③上腺素能a、β受体调控、抗心律失常、降压和抗血小板聚集等心血管作用进行了综述。阐述了某些化合物的作用机理和构效关系,还介绍了异喹啉类化合物的三种制备法,     

甲基黄酮醇胺对离体豚鼠气管的作用

本文比较性研究甲基黄酮醇胺盐酸盐（ＭＦＡ），三氟拉嗪（Ｔｒｉ），维拉帕米（Ｖｅｒ），氨茶碱（Ａｍｉ）和异丙肾上腺素（Ｉｓｏ）对豚鼠离体气管的舒张作用．ＭＦＡ（０．１－０．３ｍｍｏｌ·Ｌ－１）时间依赖性舒张ＫＣｌ诱导的气管平滑肌收缩。低于０．１ｍｍｏｌ·Ｌ－１时ＭＦＡ对ＫＣｌ诱导的收缩仅有轻微作用，却使Ｉｓｏ和Ａｍｉ的浓度舒张曲线明显向左上移动。ＭＦＡ，Ｖｅｒ，Ｔｒｉ使ＣａＣｌ２量效曲线向右下移，ｐＤ２值分别为：５．０±ｓ０．５，６．４±ｓ０．８，５．０±ｓ０．６．ＭＦＡ，Ｖｅｒ，Ｔｒｉ，Ａｍｉ剂量依赖性抑制次大量生理激动剂所致的收缩，其作用强度顺序为：Ｔｒｉ＞Ｖｅｒ＝ＭＦＡ＞Ａｍｉ．提示ＭＦＡ具有非竞争性钙拮抗作用，其作用方式与Ｔｒｉ相似。与Ａｍｉ相比、ＭＦＡ是一个较强的主气管扩张剂，并与Ａｍｉ，Ｉｓｏ具有明显的协同舒张支气管作用。     

API_（0134）对钙调素及其依赖性环核苷酸磷酸二酯酶的作用

应用钙调素（ＣａＭ）靶酶环核苷酸磷酸二酯酶（ＰＤＥ－Ｉ）活性测定方法观察穿心莲有效成分（ＡＰＩ０１３４）拮抗ＣａＭ的作用。当ＡＰＩ０１３４浓度大于７．８ｍｇ·Ｌ－１时能明显抑制ＣａＭ激活ＰＤＥ－Ｉ的活性，其半抑制浓度（ＩＣ５０）为２６·９ｍｇ·Ｌ－１，在浓度为７．８～１２５ｍｇ·Ｌ－１范围内，ＡＰＩ０１３４对ＰＤＥ－Ｉ的基础活性没有影响。     

粉防己碱抗纤维化机理研究进展

粉防己碱 (Ｔｅｔｒａｎｄｒｉｎｅ，ＴＥＴ)异名汉防己碱、汉防己甲素 ,是从防己科植物粉防己 (ＳｔｅｐｈａｎｉａＴｅｔｒａｎｄｒａＳ Ｍｏｏｒｅ)的干燥根中提取的一种双苄基异喹啉类生物碱［１］,作用广泛。近来 ,其抗纤维化作用在临床上开始受到重视 ,已在肝、肺疾病治疗中得到应用 ,并由此推广到对体表创伤后瘢痕的防治。随着细胞分离、培养技术的发展及分子生物学的应用 ,使得从基因分子水平阐述ＴＥＴ抗纤维化作用机制成为可能。１钙拮抗作用细胞浆Ｃａ２ + 浓度升高是多种疾病的病理生理基础。研究表明 ,胞浆内游离Ｃａ２ + 可通…     

Interaction of berbamine compound E6 and calmodulin-dependent myosin light chain kinase.

The interaction of the berbamine compound E6 and calmodulin (CaM)-dependent myosin light chain kinase (MLCK) has been studied. The experimental results showed that the inhibition of MLCK activity was increased with increasing amounts of E6 and was overcome completely by the addition of excessive CaM. The stimulatory activity of MLCK induced by CaM was gradually inhibited by the increasing concentrations of compound E6, showing that the inhibition of MLCK activity by compound E6 was concentration dependent; and the Ki was 0.95 microM. Compound E6 diminished the fluorescence intensity of dansyl-labeled CaM and the intensity was increased gradually by the addition of different amounts of CaM. Compound E6 had no effect on the activity of MLCK fragments produced by limited trypsinization, and it is a novel and considerably potent calmodulin antagonist.     

Distinct mechanisms of inhibition of purified cardiac sarcolemma Ca(2+)-ATPase by two calmodulin antagonists.

The effects of calmidazolium and compound 48/80 were studied in four different states of activation of the purified Ca(2+)-ATPase from cardiac sarcolemma: "basal" or unactivated, activated by calmodulin, activated by phosphatidylserine, and activated by controlled trypsinization. When assayed in the presence of phosphatidylcholine as the sole phospholipid (basal state), the purified enzyme was resistant to inhibition by calmidazolium (0.1 to 3 microM). In the same range, calmidazolium inhibited the enzyme activated by controlled proteolysis as well as the calmodulin-activated enzyme regardless of the calmodulin concentration. The phosphatidylserine-activated enzyme was inhibited at higher calmidazolium concentrations due to non-specific trapping of the inhibitor by the excess of phospholipid. Addition of calmidazolium did not modify the K0.5 for calcium activation of ATP hydrolysis by the enzyme. The inhibition by calmidazolium was counteracted by Pi. Compound 48/80 also had no effect on the enzyme when only phosphatidylcholine was present and, like calmidazolium, it inhibited the calmodulin-activated enzyme and the phosphatidylserine-activated enzyme. The apparent Ki for inhibition by compound 48/80 was dependent on the calmodulin concentration. However, the enzyme activated by controlled trypsinization was insensitive to compound 48/80. Binding of 48/80 to the enzyme in the presence of phosphatidylserine or calmodulin reversed the increased affinity for Ca2+ caused by these activators.     

Persistent alterations of calmodulin kinase II activity in chickens after an oral dose of tri-o-cresyl phosphate.

Calmodulin kinase II has been found to be involved in the increased phosphorylation of brain microtubule and spinal cord neurofilament triplet proteins following treatment of animals with organophosphorus compounds that are capable of producing organophosphorus compound-induced delayed neurotoxicity (OPIDN). In this report, chickens were given a single oral neurotoxic dose of 750 mg/kg tri-o-cresyl phosphate (TOCP), and killed after 1 or 21 days of treatment. Crude calmodulin kinase II from brain cytosol as well as phosphocellulose-purified microtubules were prepared from control and treated animals. Phosphorylation reactions were started by adding protein into the phosphorylation buffer in the presence of Mg2+, Ca2+, calmodulin or trifluoperazine, and [gamma-32P]ATP. Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subjected to autoradiography. The extent of the calmodulin kinase II autophosphorylation as well as the Ca2+/calmodulin-dependent phosphorylation of the purified microtubules was investigated. The enzyme activities isolated from control and treated animals were compared. Autophosphorylation of calmodulin kinase II was found to be higher in both 1-day and 21-day TOCP-treated animals than in control animals. The activity of the kinase to phosphorylate exogenous substrates such as tubulin and microtubule-associated protein-2 (MAP-2) was also higher in the treated hens than in the controls. The increased activity of the kinase was noted at day 1 following treatment when no clinical signs were observed and persisted until day 21 when the animals were paralyzed completely. This finding supports the significance of altered calmodulin kinase II in the pathogenesis of OPIDN.     

Radiolabelled calmodulin ligands: their low affinity and high lipophilicity may lead to artefacts in binding studies.

In order to characterize the interaction site of a series of putative calmodulin antagonists of the diphenylalkylamine type with calmodulin (CaM), a representative member of this chemical class was radiolabelled. The binding of the selected compound, [3H]-VUF 4576, to calmodulin was studied according to a recently described technique using CaM agarose. However, some peculiar results were obtained: the tight binding of [3H]-VUF 4576 increased in presence of cold VUF 4576, resulting in a high non-specific binding. The unexpected results could readily be explained by a high binding capacity of the labelled compound and the cold ligands to the walls of the test tubes used. Such results were also found when [3H]-chlorpromazine ([3H]-CPZ) was applied. In literature comparable findings have been published. To explain such results the influence of positive cooperativity or irreversible binding has been suggested. We suppose that not only in our study, but also in other published investigations, binding to glass of the radioligand and/or the cold compounds may have had a strong influence. We suggest, therefore, that care should be taken in interpreting non-classical displacement data obtained with ligands which combine a rather low affinity and a high degree of lipophilicity, not only for binding to calmodulin, but for other systems as well.     

Inhibition by trifluoperazine of calmodulin-induced activation of ATPase activity of rat erythrocyte

An endogenous, heat-stable, calcium binding protein (calmodulin). which was previously shown to increase the activity of one of the forms of cyclic nucleotide phosphodiesterase, was found to increase selectively the activity of a (Ca²⁺ + Mg²⁺)-ATPase of rat erythrocyte membranes. The ED₅₀ for calmodulin activation was 150 ng calmodulin/ml. The concentration of Ca²⁺ required for half-maximum calmodulin-induced activation of erythrocyte ATPase was 20 μM whereas approximately 50 μM Ca²⁺ was required for half-maximum calcium-induced activation of ATPase measured in the absence of calmodulin.The phenothaizine trifluoperazine, which specifically inhibits the activation of phosphodiesterase by high-affinity, calcium-specific binding to calmodulin, specifically inhibited the calmodulin-induced acti- vation of ATPase; the I₅₀ for inhibition of ATPase was 50 μM when measured in the presence of calmodulin but was over 250 μM when measured in its absence. This trifluoperazine-induced inhibition of ATPase could be overcome by adding excess calmodulin.These results indicate that calmodulin activates a specific form of erythrocyte ATPase and that trifluoperazine selectivity inhibits this activation presumably by binding to calmodulin. The results further support the hypothesis that several of the biochemical actions of phenothiazine antipsychotics can be explained by a common mechanism, namely, by selectively binding to calmodulin and thereby inhibiting its action.     

Gossypol inhibits calcineurin phosphatase activity at multiple sites

Calcineurin, the Ca2+/calmodulin-dependant serine/threonine phosphatase is the target for the immunosuppressant drugs FK506 and cyclosporine-A. These established calcineurin inhibitors each require an immunophilin protein cofactor. Gossypol, a polyphenol produced by the cotton plant, inhibits calcineurin (IC50=15 microM), in a noncompetitive, reversible manner, and is independent of any cofactor. We found that gossypol acts by at least two mechanisms to inhibit calcineurin phosphatase activity. A calmodulin-independent form of calcineurin was less sensitive to inhibition by gossypol than native calcineurin (IC50=41 and 18 microM, respectively) indicating that gossypol may interfere with calmodulin binding. A fluorescence polarization based assay demonstrated that 100 microM gossypol reduced the affinity of calmodulin for calcineurin (from K(d)=2.4 to 250 nM). Inhibition of calcineurin phosphatase activity by gossypol could not be overcome by adding excess calmodulin or by testing the inhibition toward a calmodulin-independent calcineurin indicating that gossypol acts at a site different from the calmodulin-binding site. Gossypol decreased the affinity of calcineurin for immunosuppressant/immunophilin complexes only in the presence of calmodulin, indicating that gossypol blocks the effects of calmodulin binding to calcineurin. In addition, gossypol had a stimulatory effect on native calcineurin in the absence of calmodulin, possibly indicating a calmodulin mimetic effect. Gossypol exists in two enantiomeric forms which are reported to have different potency for cell toxicity. (+) and (-) gossypol had equivalent potency for inhibition of native and calmodulin-independent calcineurin phosphatase activity, and for inhibition of calmodulin binding. The inhibition of calcineurin by gossypol via multiple binding sites without stereo-specificity indicates that gossypol is not a specific calcineurin inhibitor.     

Influence of CGS 9343B, an inhibitor of calmodulin activity, on histamine release from isolated rat mast cells.

Investigations of calmodulin involvement in cell responses has been complicated by the lack of selective calmodulin antagonists. A novel inhibitor, CGS 9343B, reportedly without influence on protein kinase C, is used in the present study of mast cell responses. The histamine release induced by antigen and compound 48/80 in the presence of calcium was enhanced by 10-20 microM CGS 9343B and inhibited by higher concentrations. Only inhibitory effects on the response to compound 48/80 in the absence of calcium and to the ionophore A23187 were observed, the latter being inhibited by 20 microM CGS 9343B. The influence on responses to combinations of the phorbol ester TPA and the ionophore A23187 was more complex, giving rise to enhancement at lower and inhibition at higher concentrations of CGS 9343B in a manner which depended on the experimental conditions. Unlike previously used calmodulin antagonists, CGS 9343B is devoid of detergent effects and without serious metabolic interference. The inhibitor seems useful to reveal differences in the mechanisms involved in responses to various histamine liberators. Our results conform with an inhibition of calmodulin by CGS 9343B but are at present inconclusive.     

Erythrocyte membrane stabilization by calcium channel blockers, calmodulin antagonists and scavengers of oxygen free radicals

Calcium channel blockers (nifedipine, verapamil, diltiazem), calmodulin antagonists (trifluoperazine, calmidazolium, compound 48/80) and anti-free radical agents (allopurinol, desferrioxamine, mannitol, L-methionine) were tested for their potency to stabilize human erythrocytes against hypotonic hemolysis. The anti-free radical agents and compound 48/80 did not confer the membrane stabilization. Nifedipine, verapamil, diltiazem, calmidazolium and trifluoperazine at low concentrations, protected the cells from the hypotonic hemolysis while at higher concentrations they caused lysis. Similar biphasic changes were produced by the detergents sodium dodecyl sulphate (SDS) and Triton X-100. The drug concentration-dependency of the biphasic changes in the erythrocytes osmotic fragility produced by calcium channel blockers and calmodulin antagonists was not affected by low concentrations of SDS and Triton X-100. On the other hand, these drugs did not prevent the hemolysis produced by high concentrations of the detergents. The above as well as the observation that the membrane stabilization is conferred only by relatively high concentrations of calcium channel blockers and calmodulin antagonists suggest that membrane stabilization is not responsible for anti-ischemic effects of these agents reported in the literature.     

6-取代氨基-1,2-双(对氯苯)-己烯-1 类化合物的设计、合成及钙调素拮抗活性的研究

对Toplis寻搜法加以改进合成5个化合物,找到6-氨基-1,2-双(对氯苯)-己烯-1化合物氨基上不同取代基的“合成终点”。用分子图形学的方法对其结构与活性的关系进行研究,结果表明药物的亲脂部分处于CaM活性部位的疏水“口袋”中,而亲水部分处于“口袋”的外部,氨基上的取代基对于药物与CaM的结合影响不大。