甲胎蛋白对HeLa细胞增殖的促进作用

为探讨甲胎蛋白 (alpha fetoprotein ,AFP)对肿瘤细胞增殖的促进作用。用从人脐带血中提取的AFP作用于体外培养的HeLa细胞 ,通过MTT计数、[3 H] TdR参入法、流式细胞仪等研究细胞增殖 ,并用放射免疫法测定细胞内cAMP、共聚焦显微镜扫描细胞内Ca2 浓度等观察细胞内第二信使物质的改变及测定3 2 P转移反映细胞内蛋白激酶A(PKA)活性 ;用Western印迹分析法分析突变型 p5 3、p2 1ras蛋白表达。结果显示AFP(浓度大于 10mg/L)对HeLa细胞增殖有明显的促进作用。在AFP(2 0mg/L)作用下 ,HeLa细胞cAMP浓度升高 30 0 % ;Ca2 浓度最大升高 15 4 .9%、PKA活性最大升高 10 0 %。突变型 p5 3和 p2 1ras分别增高 81.1% (2 4h)和 96 .2 % (2 4h)。抗甲胎蛋白单克隆抗体能有效地阻断AFP促HeLa细胞增殖作用。以上表明 ,(1)AFP具促HeLa细胞增殖的生理功能 ;(2 )AFP促进HeLa细胞增殖的作用是通过影响细胞内cAMP和Ca2 浓度以及PKA活性进而促进一些原癌基因的表达来实现的。     

RhoA蛋白和cAMP对人前列腺癌细胞株PC-3形态的影响(简报)

小G蛋白RhoA是细胞内信号转导的重要成分,参与对细胞的多种功能活动的调控。溶血磷脂酸(lysophosphatidicacid,LPA)与多种细胞的G蛋白偶连受体结合而发挥作用,除刺激细胞增殖外,还通过活化RhoA,诱导细胞骨架改变。cAMP是经典的第二信使,其下游激酶PKA可抑制RhoA活性,因此,cAMP在许多细胞活动中对RhoA有拮抗作用。本实验采用人前列腺癌细胞株PC-3,以绿色荧光蛋白(GreenFluorescentProtein,GFP)分别和不同RhoA结构(野生型RhoA、RhoA63L和RhoA188A)的cDNA共同转染细胞,在显微镜下(200倍视野)观察记录未转染细胞和转染细胞在LPA和cAMP作用下的形态变化,研究RhoA和cAMP/PKA介导的信号转导在调控癌细胞形态改变中的作用。     

白介素-2对心肌细胞[Ca~(2 )]_i的作用及其信号转导途径

为研究白介素 2 (interleukin 2 ,IL 2 )对心肌细胞内钙浓度 ([Ca2 ]i)的影响及其信号转导途径 ,实验采用酶解法分离成年大鼠心室肌细胞 ,以Fura 2 /AM为钙探针 ,用细胞内双波长钙荧光系统检测细胞 [Ca2 ]i 的变化。结果发现 :(1)IL 2 (0 5～ 2 0 0U/ml)浓度依赖性地降低单个心室肌细胞内钙瞬态 ,IL 2 (2 0 0U/ml)对咖啡因诱导的肌浆网内储钙的释放无影响 ;(2 )纳洛酮 (naloxone ,Nal) (10 -8mol/L)和nor binaltorphimine (nor BNI,10 -8mol/L)可阻断IL 2对心肌细胞钙瞬态的作用 ,而纳曲吲哚 (naltrindole ,NTI) (10 -6mol/L)不能阻断此作用 ;(3)κ阿片受体激动剂U5 0 488H (10 -6mol/L)降低心肌细胞钙瞬态 ,nor BNI (10 -8mol/L)可阻断此作用 ;(4 ) 5mg/L百日咳毒素 (PTX)预处理可取消IL 2降低心肌细胞钙瞬态的作用 ,而酪氨酸激酶抑制剂genistein (10 -4 mol/L)不能取消IL 2的作用 ;(5 )U7312 2预处理可阻断IL 2的作用。研究结果表明 ,IL 2降低心肌细胞钙瞬态的作用 ,是通过心肌细胞上κ阿片受体介导的 ,其下游途径包括PTX敏感的G蛋白和磷脂酶C。     

甲硫氨酸脑啡肽和抗δ阿片受体抗体对小鼠骨髓瘤细胞信号传递系统的作用

为了探讨阿片肽与细胞表面受体结合后所产生的生物效应及其机制 ,用不同浓度甲硫氨酸脑啡肽 ( MENK)及抗 δ阿片受体单克隆抗体处理小鼠的骨髓瘤细胞 ( NS- 1 ) ,然后测定蛋白激酶A( PKA) ,蛋白激酶 C( PKC)活性及三磷酸肌醇 ( IP3 )含量 .研究结果表明 ,NENK可升高细胞胞浆及胞膜 PKA的活性 ,且这一作用可被抗δ阿片受体抗体所拮抗 .MENK对 PKC影响呈双向反应 ,0 .1 μmol/L MENK可以升高胞浆 PKC活性 ,但却明显降低胞膜 PKC活性 ;在 MENK浓度为 1 0μmol/L时则情况刚好相反 .1 μmol/L的 MENK可明显降低胞浆及胞膜 PKC活性 ,抗体可拮抗这种下调作用 .MENK可降低细胞内 IP3 的含量 ,且这一作用可被抗δ阿片受体抗体所拮抗 .由此可以推论 :MENK在与 δ阿片受体结合后 ,可以经过多种信号传导系统来调节细胞功能 ,从而产生不同的生物效应 .     

大鼠心肌肌浆网和核被膜ryanodine受体动力学特性的比较

目的 :观察大鼠心肌浆网 (sarcoplasmicreticulum ,SR)和核被膜 (nuclearenvelope ,NE)ryanodine受体 (RyR)与配体结合特点及其蛋白质磷酸化调节。方法 :采用差速和等密度梯度离心分离心肌SR和NE ,用放射受体分析法研究RyR的特征。结果 :NE上存在高亲和力RyR ,其最大结合 (Bmax)为SRRyR的 1.7% ,解离常数 (Kd)为SR的6 0 %。分别用PKA和PKC磷酸化后 ,SR上该受体的Bmax各增加 3.7和 1.2倍 ,而NE上的该受体Bmax各增加 2 .2和 3.1倍 ,Kd均无显著改变。结论 :NE上存在比SR密度低但亲和力高的RyR ,能被PKA和PKC激活 ,而且对PKC较PKA更敏感     

RhoA蛋白和cAMP对人前列腺癌细胞株PC-3形态的影响（简报）

小G蛋白RhoA是细胞内信号转导的重要成分,参与对细胞的多种功能活动的调控。溶血磷脂酸（lysophosphatidic acid,LPA）与多种细胞的G蛋白偶连受体结合而发挥作用,除刺激细胞增殖外,还通过活化RhoA,诱导细胞骨架改变。cAMP是经典的第二信使,其下游激酶PKA可抑制RhoA活性,因此,cAMP在许多细胞活动中对RhoA有拮抗作用。     

甲胎蛋白对HeLa细胞N-ras、p53和p21~(ras)表达的促进作用

大量研究已证明甲胎蛋白 (alpha fetoprotein ,AFP)对肿瘤细胞的增殖具有调节作用 .为探讨AFP对细胞生长促进作用的分子机理 ,采用从人脐带血中提取的AFP作用于体外培养的HeLa细胞 ,用Northern印迹分析法分析不同作用时间时细胞N rasmRNA的表达以及用Western印迹分析法分析p5 3、p2 1ras的表达 .结果发现 ,在AFP(2 0mg L)作用后 ,HeLa细胞的N rasmRNA、p5 3蛋白质和p2 1ras蛋白质的表达量与对照组比较在 12h和 2 4h时都有明显增加 .AFP的作用均可被抗AFP单克隆抗体所拮抗 .实验结果提示 ,AFP对细胞生长的调节作用可能通过促进这些原癌基因的表达来实现 .     

aFGF对人脐静脉内皮细胞TPK、PKC活性及Ca~(2+)浓度的影响

为了观察酸性成纤维细胞生长因子 ( acidic fibroblast growth factor,a FGF)与人脐静脉内皮细胞 ( human umbilical vein endothelial cell,HUVEC)膜上特异受体结合后引起的细胞内信号转导途径 ,探讨 a FGF导致细胞增殖的机理 ,经 Scatchard曲线分析人脐静脉内皮细胞膜受体性质 .以不同浓度的 a FGF处理人脐静脉内皮细胞 ,利用 [γ- 3 2 P]ATP参入外源性底物的方法测定受体的酪氨酸蛋白激酶 ( tyrosine protein kinase,TPK)及蛋白激酶 C( protein kinase C,PKC)的活性 ;用 Fura-2 /AM为荧光指示剂测定 [Ca2 + ]i.结果显示 :Scatchard曲线证明 a FGF与 HUVEC膜受体特异结合呈一条曲线 ,即受体为一种结合位点 ,Kd 为 3.6× 1 0 -10～ 9.6× 1 0 -10 mol/L,每个细胞受体数为2 70 90 .随着 a FGF浓度增加 ,TPK及 PKC活性随之升高 .当 a FGF浓度为 1 .1 2 mg/L时 ,a FGF处理组的 TPK活性是对照组的 3倍 ;膜 PKC活性是对照组 3.4倍 ,胞浆 PKC活性是对照组的 1 .87倍 .胞浆 [Ca2 + ]是对照组的 3倍 .结果指出 :该细胞中 a FGF受体具有 TPK活性 .TPK激活后进一步促进蛋白质和酶磷酸化级联反应 ,而使 PKC活性及 [Ca2 + ]i 升高 ,即 PKC和 Ca2 + 为 TPK的下游信号分子 ,进一步促进基因表达增加 ,导致细胞增殖 .     

aFGF对人脐静脉内皮细胞TPK、PKC活性及Ca2+浓度的影响

为了观察酸性成纤维细胞生长因子 ( acidic fibroblast growth factor,a FGF)与人脐静脉内皮细胞 ( human umbilical vein endothelial cell,HUVEC)膜上特异受体结合后引起的细胞内信号转导途径 ,探讨 a FGF导致细胞增殖的机理 ,经 Scatchard曲线分析人脐静脉内皮细胞膜受体性质 .以不同浓度的 a FGF处理人脐静脉内皮细胞 ,利用 [γ- 3 2 P]ATP参入外源性底物的方法测定受体的酪氨酸蛋白激酶 ( tyrosine protein kinase,TPK)及蛋白激酶 C( protein kinase C,PKC)的活性 ;用 Fura-2 /AM为荧光指示剂测定 [Ca2 ]i.结果显示 :Scatchard曲线证明 a FGF与 HUVEC膜受体特异结合呈一条曲线 ,即受体为一种结合位点 ,Kd 为 3.6× 1 0 -10～ 9.6× 1 0 -10 mol/L,每个细胞受体数为2 70 90 .随着 a FGF浓度增加 ,TPK及 PKC活性随之升高 .当 a FGF浓度为 1 .1 2 mg/L时 ,a FGF处理组的 TPK活性是对照组的 3倍 ;膜 PKC活性是对照组 3.4倍 ,胞浆 PKC活性是对照组的 1 .87倍 .胞浆 [Ca2 ]是对照组的 3倍 .结果指出 :该细胞中 a FGF受体具有 TPK活性 .TPK激活后进一步促进蛋白质和酶磷酸化级联反应 ,而使 PKC活性及 [Ca2 ]i 升高 ,即 PKC和 Ca2 为 TPK的下游信号分子 ,进一步促进基因表达增加 ,导致细胞增殖 .     

大豆异黄酮对大鼠乳腺癌细胞内cAMP/PKA信号途径的影响

本实验研究了大豆异黄酮对SHZ-88大鼠乳腺癌细胞内cAMP／PKA信号途径的影响。实验设3组：空白对照组、50μg／ml大豆黄酮及15μg／ml染料木素组。采用放射免疫测定法（RIA）检测了胞内cAMP的浓度、腺苷酸环化酶（adenylate cyclase,AC）和磷酸二酯酶（phosphodiesterase,PDE）的活性,用（γ-^32P）ATP掺入法测定cAMP依赖性PKA的活性,半定量RT-PCR法分析cAMP反应元件结合蛋白（cAMP response element binding protein,CREB）mRNA表达的变化。结果表明：在处理后5min,大豆黄酮组和染料木素组细胞的cAMP浓度分别比对照组升高了9．5％和11．0％（P〈0．05）：10min时,分别比对照组升高31．0％和40.3％（P〈0．01）。3组细胞的AC活性在处理时间内没有明显变化。但在处理后5min,大豆黄酮组和染料木素组细胞的PDE活性分别降至对照组的71．8％和71．6％（P〈0．05）。处理后20min,大豆黄酮组和染料木素组细胞PKA活性分别上升到对照组的125．8％和122．3％（P〈0．05）;到40min时仍维持在高水平。大豆黄酮组和染料木素组细胞CREB mRNA的表达量在处理后3h分别比对照组增加31．6％和51．1％（P〈0．05）;6h后开始下降。这些结果提示,大豆异黄酮能够激活大鼠乳腺癌细胞内cAMP／PKA信号途径;而且是通过抑制磷酸二酯酶的活性,导致胞内cAMP浓度升高而实现的。     

The intracellular mechanism of alpha-fetoprotein promoting the proliferation of NIH 3T3 cells

AIM The existence and properties of alpha-fetoprotein (AFP) receptor on the surface of NIH 3T3 cells and the effects of AFP on cellular signal transduction pathway were investigated. METHODS The effect of AFP on the proliferation of NIH 3T3 cells was measured by incorporation of 3H-TdR. Receptor-binding assay of 125I-AFP was performed to detect the properties of AFP receptor in NIH 3T3 cells. The influences of AFP on the [cAMP]i and the activities of protein kinase A (PKA) were determined. Western blot was used to detect the change of K-ras P21 protein expression. RESULTS The proliferation of NIH 3T3 cells treated with 0-80 mg/L of AFP was significantly enhanced. The Scatchard analysis indicated that there were two classes of binding sites with KD of 2.722×10-9M (Bmax=12810 sites per cell) and 8.931× 10-8M (Bmax=119700 sites per cell) respectively. In the presence of AFP (20 mg/L), the content of cAMP and activities of PKA were significantly elevated . The level of K-ras P21 protein was upregulated by     

Transmodulation of epidermal growth factor receptor function by cyclic AMP-dependent protein kinase.

Binding of epidermal growth factor (EGF) to its receptor (EGFR) augments the tyrosine kinase activity of the receptor and autophosphorylation. Exposure of some tissues and cells to EGF also stimulates adenylyl cyclase activity and results in an increase in cyclic AMP (cAMP) levels. Because cAMP activates the cAMP-dependent protein kinase A (PKA), we investigated the effect of PKA on the EGFR. The purified catalytic subunit of PKA (PKAc) stoichiometrically phosphorylated the purified full-length wild type (WT) and kinase negative (K721M) forms of the EGFR. PKAc phosphorylated both WT-EGFR as well as a mutant truncated form of EGFR (Delta1022-1186) exclusively on serine residues. Moreover, PKAc also phosphorylated the cytosolic domain of the EGFR (EGFRKD). Phosphorylation of the purified WT as well as EGFRDelta1022-1186 and EGFRKD was accompanied by decreased autophosphorylation and diminished tyrosine kinase activity. Pretreatment of REF-52 cells with the nonhydrolyzable cAMP analog, 8-(4-chlorophenylthio)-cAMP, decreased EGF-induced tyrosine phosphorylation of cellular proteins as well as activation of the WT-EGFR. Similar effects were also observed in B82L cells transfected to express the Delta1022-1186 form of EGFR. Furthermore, activation of PKAc in intact cells resulted in serine phosphorylation of the EGFR. The decreased phosphorylation of cellular proteins and diminished activation of the EGFR in cells treated with the cAMP analog was not the result of altered binding of EGF to its receptors or changes in receptor internalization. Therefore, we conclude that PKA phosphorylates the EGFR on Ser residues and decreases its tyrosine kinase activity and signal transduction both in vitro and in vivo.     

MAP kinase activation by mu opioid receptor involves phosphatidylinositol 3-kinase but not the cAMP/PKA pathway.

The involvement of protein kinases was studied in mu opioid receptor activation of mitogen-activated protein (MAP) kinase using cells transfected with the receptor clone. The cAMP/protein kinase A (PKA) pathway is known to be the major biochemical pathway for mu opioid receptor signaling. However, our data showed that stimulating adenylyl cyclase or activating PKA had no effect on mu receptor enhancement of MAP kinase activity, suggesting that the cAMP/PKA pathway is not involved in mediating the mu receptor activation of MAP kinase. Inhibition of phosphatidylinositol (PI) 3-kinase reduced mu receptor enhancement of MAP kinase activity, suggesting PI 3-kinase involvement. Together, these results show that cross-talk between the mu opioid receptor and the MAP kinase cascade is not mediated by the cAMP/PKA pathway, but involves PI 3-kinase.     

Melatonin receptor signaling in pregnant and nonpregnant rat uterine myocytes as probed by large conductance Ca2+-activated K+ channel activity

The mRNAs of MT1 and MT2 melatonin receptors are present in cells from nonpregnant (NPM) and pregnant (PM) rat myometrium. To investigate the coupling of melatonin receptors to Gq- and Gi-type of heterotrimeric G proteins, we analyzed the activity of large-conductance Ca2+-activated K+ (BKCa) channels, the expression of which in the uterus is confined to smooth muscle cells. The melatonin receptor agonist 2-iodomelatonin induced a pertussis toxin (PTX)-insensitive increase in channel open probability that was blocked by the nonselective antagonist luzindole. The 2-iodomelatonin effect on channel open probability was suppressed by overexpression of the Gqalpha-inactivating protein RGS16 and the phospholipase C inhibitor U-73122. The activity of BKCa channels is differentially regulated by protein kinase A (PKA) in NPM and PM cells. Thus, the beta-adrenoceptor agonist isoprenaline inhibited the BKCa channel conducted whole-cell outward current (Iout) in NPM cells and enhanced Iout in PM cells. Additional application of 2-iodomelatonin antagonized the isoprenaline effect on Iout in NPM cells but enhanced Iout in PM cells. All 2-iodomelatonin effects on Iout were sensitive to PTX treatment and the PKA inhibitor H-89. We therefore conclude that melatonin activates both the PTX-insensitive Gq/phospholipase C/Ca2+ and the PTX-sensitive Gi/cAMP/PKA signaling pathway in rat myometrium.     

Apoptotic signaling through the beta -adrenergic receptor. A new Gs effector pathway

Stimulation of beta-adrenergic receptor normally results in signaling by the heterotrimeric G protein G(s), leading to the activation of adenylyl cyclase, production of cAMP, and activation of cAMP-dependent protein kinase (PKA). Here we report that cell death of thymocytes can be induced after stimulation of beta-adrenergic receptor, or by addition of exogenous cAMP. Apoptotic cell death in both cases was observed with the appearance of terminal deoxynucleotidyl transferase-mediated UTP end labeling reactivity and the activation of caspase-3 in S49 T cells. Using thymocytes deficient in either Galpha(s) or PKA, we find that engagement of beta-adrenergic receptors initiated a Galpha(s)-dependent, PKA-independent pathway leading to apoptosis. This alternative pathway involves Src family tyrosine kinase Lck. Furthermore, we show that Lck protein kinase activity can be directly stimulated by purified Galpha(s). Our data reveal a new signaling pathway for Galpha(s), distinct from the classical PKA pathway, that accounts for the apoptotic action of beta-adrenergic receptors.     

Phosphodiesterases and compartmentalized cAMP signalling in the heart

A key feature of the cAMP/cAMP-dependent protein kinase (PKA) transduction system is the compartmentalisation of its signalling enzymes and effectors. Given the large diversity of PKA targets within cardiac cells a precisely regulated and confined activity of such signalling pathway is essential for specificity of response. This appears to be achieved through the generation of local pools of high cAMP and activation of PKA at discrete subcellular locations. Phosphodiesterases (PDEs) are the only route for degrading cAMP and are thus poised to regulate intracellular cAMP gradients. Their spatial confinement to discrete compartments and functional coupling to individual receptors provides an efficient way to control local [cAMP](i) in a stimulus-specific manner. A better understanding of the distinctive role that individual PDEs play in shaping the cAMP signal in heart cells may lead to the development of new strategies for selective pharmacologic manipulation of cAMP signalling in defined functional domains.     

Cyclic AMP-independent activation of protein kinase A by vasoactive peptides

Protein kinase A (PKA) is an important effector enzyme commonly activated by cAMP. The present study focuses on our finding that the vasoactive peptide endothelin-1 (ET1), whose signaling is not coupled to cAMP production, stimulates PKA in two independent cellular models. Using an in vivo assay for PKA activity, we found that ET1 stimulated PKA in HeLa cells overexpressing ET1 receptors and in aortic smooth muscle cells expressing endogenous levels of ET1 receptors. In these cell models, ET1 did not stimulate cAMP production, indicating a novel mechanism for PKA activation. The ET1-induced activation of PKA was found to be dependent on the degradation of inhibitor of kappaB, which was previously reported to bind and inhibit PKA. ET1 potently stimulated the nuclear factor-kappaB pathway, and this effect was inhibited by overexpression of the inhibitor of kappaB dominant negative mutant (IkappaBalpham) and by treatment with the proteasome inhibitor MG-132. Importantly, IkappaBalpham and MG-132 had similar inhibitory effects on ET1-induced activation of PKA without affecting G(s)-mediated activation of PKA or ET1-induced phosphorylation of mitogen-activated protein kinase. Finally, another vasoactive peptide, angiotensin II, also stimulated PKA in a cAMP-independent manner in aortic smooth muscle cells. These findings suggest that cAMP-independent activation of PKA might be a general response to vasoactive peptides.     

Modulation of NMDA receptor function by cyclic AMP in cerebellar neurones in culture

The signal transduction pathways involved in NMDA receptor modulation by other receptors remain unclear. cAMP could be involved in this modulation. The aim of this work was to analyse the contribution of cAMP to NMDA receptor modulation in cerebellar neurones in culture. Forskolin increases cAMP and results in increased intracellular calcium and cGMP that are prevented by blocking NMDA receptors. Similar effects were induced by two cAMP analogues, indicating that cAMP leads to NMDA receptor activation. It has been reported that phosphorylation of Ser897 of the NR1 subunit of NMDA receptors by cAMP-dependent protein kinase (PKA) activates the receptors. Forskolin increases Ser897 phosphorylation. Neither Ser897 phosphorylation nor cGMP increase induced by forskolin are prevented by four inhibitors of PKA, suggesting that NMDA receptor activation is dependent on cAMP but not on PKA. Inhibition of Akt prevents forskolin-induced phosphorylation of Ser897, suggesting a role for Akt in the mediation of the modulation of NMDA receptors by cAMP. Pituitary adenylate cyclase-activating polypeptide (PACAP) activates its receptors, increasing cAMP and also leading to phosphorylation of Ser897 of NR1 and activation of NMDA receptors. These results indicate that cAMP modulates NMDA receptor in cerebellar neurones and may play a role in NMDA receptor modulation by other receptors.