钙调神经磷酸酶信号通路参与钙激动剂介导的心肌细胞肥大

目的在于探讨不同来源的细胞内游离钙(［Ca2+］i）对钙调神经磷酸酶（calcineurin,CaN）依赖信号通路和心肌细胞肥大的影响。方法以原代培养的乳鼠心肌细胞为模型,血管紧张素Ⅱ（AngⅡ）、雷尼丁（RY）和三磷酸肌醇（IP3）（浓度均为10-7mol／L）激活心肌细胞［Ca2+］i,应用钙荧光指剂Fura-2／AM动态观测心肌细胞［Ca2+］i浓度,同时检测心肌细胞CaN活性及蛋白表达。用氚-亮氨酸（3H-Leu）掺入量测定心肌细胞蛋白质合成速率。结果发现AngⅡ、RY和IP3明显增加心肌细胞［Ca2+］i浓度、CaN活性及蛋白表达并提高3H-Leu的掺入量,与对照组心肌细胞相比差异显著（P＜0．01）。结论激活心肌细胞［Ca2+］i可明显提高心肌细胞蛋白合成速率,心肌细胞CaN活性及蛋白表达似与细胞［Ca2+］i变化有明显关系而与其来源无关,表明CaN信号通路在心肌细胞生长中发挥重要作用。     

钙离子,钙拮抗剂与缺血性心律失常

心肌缺血造成细胞膜系统对Ca~(2 )转运失常,导致细胞浆游离Ca~(2 )浓度提高,通过对细胞膜多种离子通道的调制作用,Ca~(2 )与缺血心肌多种病理性电活动有关,这可能是缺血性心律失常的机制之一;而钙拮抗剂通过抑制电压依赖性钙通道,减轻钙超载,从而对抗Ca~(2 )中介的缺血性心律失常。     

钙拮抗剂对呼吸跃变后苹果组织乙烯生成的影响（简报）

异博定、Ｌa^3 和Ca^2 对呼吸变后苹果（品种辽伏）圆片的乙烯生成影响不显著,高浓度钌红（２mmol.L^-1）显著抑制乙烯生成,而较低浓度的钌红（０．２５mmol.L^-1）则促进ＡＣＣ向乙烯的转化。     

果蝇钙调蛋白和肌球蛋白NINAC相互作用分析

果蝇的视觉信号转导途径是已知的最快的G 蛋白偶联信号通路。这其中涉及到TRP/TRPL通道的开放以及钙离子的内流等一系列反应的形成。NINAC（neither inactivation nor afterpotential C）是一种特异性存在于果蝇感光细胞中的第3类肌球蛋白（Myosin III）,其在终止果蝇的视觉信号转导通路中起着非常重要的作用。NINAC蛋白具有两种亚型：一种是132 kD的蛋白亚型 (p132),另一种则是174 kD的蛋白亚型(p174)。这两种不同的蛋白亚型都具有相同的激酶催化结构域(kinase domain),以及与肌球蛋白相似的马达结构域(motor domain)。但是,它们在C末端却存在着非常大的差异,这其中包括了钙调蛋白结合基序(IQ motif)。NINAC的这两种蛋白亚型在果蝇的感光细胞中的定位以及作用有很大不同,尤其是在与钙调蛋白的相互作用方面。钙调蛋白结合基序与钙调蛋白（CaM）之间的相互作用对于果蝇的视觉信号通路具有重要的意义：NINAC结合钙调蛋白能力的缺失将导致果蝇的视觉传导缺陷。本文通过蛋白共表达的方法,成功表达并纯化得到了不同版本的NINAC与钙调蛋白的蛋白复合物。静态光散射的结果表明,在Ca2+存在情况下,p174蛋白可以结合2个Ca2+-CaM,而p132只结合1个Ca2+-CaM。通过分析型凝胶过滤以及等温量热滴定技术,进一步鉴定了p174及p132的IQ2（第2个钙调蛋白结合基序）序列与Ca2+ CaM的相互作用。通过序列分析及进一步的突变实验发现,p174 IQ2中的3个疏水氨基酸（F1083,F1086 和 L1092）对于钙调蛋白的结合非常重要,并导致了p174与p132蛋白和Ca2+ CaM结合能力的差异。本文的研究提供了NINAC与Ca2+-CaM相互作用的生化机制,将为进一步在果蝇视觉信号通路中深入研究CaM是如何调节NINAC的体内功能实验打下基础。     

铝与钙调蛋白相互作用的荧光光谱研究

本文研究了铝与钙调蛋白相互作用的荧光光谱。实验证明,Al~3与CaM的结合所引起的构象变化与Ca~(2+)与CaM结合所引起的构象变化既有相同之处,也有不同之处。Al~(3+)在CaM分子上的结合有特异性结合与非特异性结合两种情况。其特异性结合位点可能为2—3个。钙调蛋白的非竞争性拮抗剂酸枣仁皂甙A(JuA)可以继续抑制已被Al~(3+)部分抑制的PDE-CaM的活力。     

蜡嘴,锡嘴雀和法国鹌鹑耳蜗—中脑听觉中枢的比较观察

用辣根过氧化物酶HRP顺行标记方法表明蜡嘴(Eophona migratoria)、锡嘴(Coccothra-ustes coccothraustes)和鹌鹑(France Coturnix coturnix)脑干内听觉中枢的初级神经元位于耳蜗核(nCO,Cochlear unclei)内。较高级神经元位于中脑背外侧核(MLD,Nucleus mesen-cephalicus lateralis,pars dorsalis)。脑干内听觉传入通路始于nCO,经外侧丘系(LL,Lemni-scus lateralis)可直接投射于MLD。鸣禽鸟蜡嘴、锡嘴是对侧投射,同侧仅有个别纤维被标记,非鸣禽鹌鹑仅是对侧性投射。     

中枢多巴胺转运蛋白的结构功能与调控

中枢多巴胺 (ＤＡ)能系统信号传递决定于突触间隙ＤＡ的浓度水平。ＤＡ在完成神经信息传递后 ,通过重摄取和酶解二种途径灭活以终止信息传递。其中大部分ＤＡ为位于突触前膜上的中枢多巴胺转运蛋白 (ＤＡＴ)所摄取 ,转运至突触前神经元以备再次利用。近年来研究发现 ,ＤＡＴ并不只是简单地重摄取ＤＡ ,ＤＡＴ同时又是调控突触间隙ＤＡ水平和维系突触前ＤＡ合成、储存功能的关键因素[1] 。另外 ,ＤＡＴ还是许多精神药物潜在的作用靶位点。因此 ,研究ＤＡＴ结构、功能及其调控 ,有助于阐明ＤＡＴ与ＤＡ系统精神神经疾病的关系和探索治疗策略。…     

鸡中枢淋巴器官肥大细胞的组织化学与形态学

对哺乳动物的,特别是啮齿动物和人类肥大细胞已有了比较深入的研究, 但关于家禽肥大细胞的研究很少.本研究旨在阐明鸡中枢淋巴器官中肥大细胞的组织化学与形态学特征.本研究证实Carnoy 氏液是鸡肥大细胞的优良的固定液,而中性缓冲福尔马林(NBF) 却阻断了大多数肥大细胞的着染力.甲苯胺蓝和阿尔新蓝是鸡肥大细胞的良好的染料,但阿尔新蓝能使更多的肥大细胞着染,虽然其也可使杯状细胞着染.作者的一种新的染色法, 长时间阿尔新蓝染色(LAB-S)可用于NBF固定的组织中肥大细胞的染色,因为其着染的细胞数与Carnoy 氏液固定甲苯胺蓝染色的细胞数无显著差异(P<0.001).在胸腺髓质中见有大量的肥大细胞,而胸腺皮质仅可见个别肥大细胞位于血管周围及小叶间结缔组织中.腔上囊的皮质与髓质中很少见有肥大细胞.肥大细胞有血管周围分布的倾向,但一个有趣的发现是血管内偶尔也有个别肥大细胞.电镜下可见肥大细胞的胞浆颗粒内充满无定形的颗粒状基质,但其电子密度有的较高,有的较低.少数胞浆颗粒内有旋涡状及网状亚微结构.但未见有人类肥大细胞胞浆颗粒内特征性的晶格状和卷轴状的亚微结构,也未见到在绵羊肥大细胞中描述过的特殊亚微结构.     

蜂毒肽片段的合成及其与钙调蛋白的相互作用

采用固相法设计合成了４个蜂毒肽片段：Ｍｅｌ１２、Ｍｅ１１３、Ｍｅｌ１４、Ｍｅｌ１５。应用电泳技术,抑制钙依赖性的磷酸二酯酶酶活方法和荧光技术研究了这些多肽与钙调蛋白的相互作用。结果表明这些多肽与钙调蛋白均形成１：１复合物,抑制钙依赖性的磷酸二酯酶的活性,其中Ｍｅｌ１４和Ｍｅｌ１５对钙调蛋白的结合活性与完整的蜂毒肽比较接近。     

Interaction of calcium agonists and antagonists with Ca-binding proteins and their effect on cyclic nucleotide phosphodiesterase

The effects of Ca2+ antagonists (nicardipine, felodipine, nitrenedipine, isradipine, niphedipine, darodipine and riodipine) and Ca2+ agonists (BAY K8644 and CGP 28392), 1.4-dihydropyridine derivatives (1.2-DHP), on the calmodulin (CM)-dependent activation of cyclic nuxleotide phosphodiesterase (PDE) were studied. Both the blockers and activators of slow potential-dependent Ca2+ channels induced a un-competitive inhibition of the CM-dependent PDE activity. 1.4-DHP was found to replace the fluorescent probe, diS-C3-(5), from the Ca2(+)-dependent calmodulin-dye complex (K0.5 = 4-60 microM) but at concentrations below 100 microM had no effect on the Ca2(+)-dependent troponin C-dye complex. Darodipine (100 microM) did not interact with the proteins. The 1.4-DHP interaction with CM did not interfere with PDE activation. It is concluded that 1.4-DHP may affect Ca2+ dependent processes not only at the levels of activation or blocking of Ca2+ channels, but also through regulation of Ca2(+)-CM dependent enzymes.     

Interaction of calcium antagonists with cyclic AMP phosphodiesterases and calmodulin

The calcium antagonists, nimodipine and nicardipine, competitively inhibited calmodulin-sensitive and calmodulin-insensitive forms of cyclic AMP phosphodiesterase, with IC_50's in the micromolar range. Verapamil showed similar inhibitory potency against calmodulin-insensitive phosphodiesterases, but in marked contrast, it was a very weak inhibitor (30–100 times less potent) against calmodulin-sensitive forms of the enzyme. Verapamil and nimodipine both antagonized the calmodulin stimulation of phosphodiesterase. Through use of hydrophobic fluorescent probes, verapamil, and another calmodulin antagonist, proadifen, were shown to interact directly with calmodulin in a manner that differed from the interaction of calmodulin with trifluoperazine.     

Vasopressin agonists and antagonists

In this article, the discrete modifications of the structure of the vasopressin molecule which led to the development of specific V1 vascular, V2 renal, and mixed V1/V2 analogs are reviewed. Particularly, the third generation of vasopressin antagonists produced by deletions and substitutions of the carboxy terminal, and the fourth generation of vasopressin antagonists obtained by deletions, substitutions, and the linearization of the molecule are presented. The potential advantages of these different compounds are illustrated by our work on V1 vascular vasopressin receptors of human platelets.     

Calcium, calcium channels, and calcium channel antagonists

Voltage-dependent Ca2+ channels are an important pathway for Ca2+ influx in excitable cells. They also represent an important site of action for a therapeutic group of agents, the Ca2+ channel antagonists. These drugs enjoy considerable use in the cardiovascular area including angina, some arrhythmias, hypertension, and peripheral vascular disorders. The voltage-dependent Ca2+ channels exist in a number of subclasses characterized by electrophysiologic, permeation, and pharmacologic criteria. The Ca2+ channel antagonists, including verapamil, nifedipine, and diltiazem, serve to characterize the L channel class. This channel class has been characterized as a pharmacologic receptor, since it possesses specific drug-binding sites for both antagonists and activators and it is regulated by homologous and heterologous influences. The Ca2+ channels of both voltage- and ligand-regulated classes are likely to continue to be major research targets for new drug design and action.     

Synthesis of novel steroidal agonists,partial agonists,and antagonists for the glucocorticoid receptor

Adverse effects of glucocorticoids could be limited by developing new compounds that selectively modulate anti-inflammatory activity of the glucocorticoid receptor (GR). We have synthesized a novel series of steroidal GR ligands, including potent agonists, partial agonists and antagonists with a wide range of effects on inhibiting secretion of interleukin-6. Some of these new ligands were designed to directly impact conformational stability of helix-12, in the GR ligand-binding domain (LBD). These compounds modulated GR activity and glucocorticoid-induced gene expression in a manner that was inversely correlated to the degree of inflammatory response. In contrast, compounds designed to directly modulate LBD epitopes outside helix-12, led to dissociated levels of GR-mediated gene expression and inflammatory response. Therefore, these new series of compounds and their derivatives will be useful to dissect the ligand-dependent features of GR signaling specificity.     

Analysis of the role of calcium in analgesic action of non-narcotic analgesics and calcium channel blockers]

In the mice hot-plate test we have compared analgesic effect of calcium channel blockers and new non-narcotic analgesic antiinflammatory agent PV-107: verapamil > fenigidin > PV-107. Simultaneously we have shown strong correlation (r - 0.82) between analgesic effect and 45Ca2+ efflux of cardiac membrane in depolarizing media in vitro.     

Interaction of charged and uncharged calcium channel antagonists with phospholipid membranes. Binding equilibrium, binding enthalpy, and membrane location.

The membrane location and the binding mechanism of two Ca2+ channel antagonists, amlodipine and nimodipine, in pure lipid membranes were investigated with deuterium and phosphorus-31 nuclear magnetic resonance, with thermodynamic methods such as high-sensitivity titration calorimetry, and by measuring the membrane surface charge via the zeta-potential. The two drugs exhibit quite different physical-chemical properties. The noncharged nimodipine is strongly hydrophobic, and selective deuteration of the lipid membrane reveals a homogeneous distribution of nimodipine across the whole hydrocarbon layer, but no interaction at the lipid headgroup level. The membrane behavior of the amiphiphilic amlodipine (electric charge z = +1) is distinctly more complex. Deuterium magnetic resonance demonstrates that amlodipine adopts a well-defined position in the bilayer membrane. In particular, the charged ethanolamine side group of amlodipine is located near the water-lipid interface, interacting with the dipoles of the headgroup region according to a nonspecific, electrostatic mechanism and inducing a reorientation of the phosphocholine dipoles toward the water phase. At the level of the hydrocarbon segment, the nonpolar ring system of amlodipine interacts specifically with the cis double bond of the membrane lipid, forming a weak association complex. With increasing amlodipine concentration the deuterium signal of the cis double bond gradually loses intensity, a phenomenon previously observed only in related studies on protein-lipid interactions. The binding equilibrium of amlodipine to phosphatidylcholine membranes was studied by measuring the electrophoretic mobility of lipid vesicles and with a centrifugation assay. Hydrophobic interactions of the nonpolar ring systems and electrostatic repulsions at the membrane surface contribute to the binding energy.(ABSTRACT TRUNCATED AT 250 WORDS)