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 .     

Epstein-Barr病毒感染对CNE-2Z不同细胞组分中PKC和TPK活性的影响

研究EBV体外再感染CNE-2Z细胞后,不同组分中PKC(蛋白激酶C)和TPK(酪氨酸蛋白激酶)活性的影响,并探讨PKC和TPK活性与细胞增殖的关系。实验分三组即对照组、EBV组和EBV+TPA组,用免疫细胞化学(以小鼠抗EB病毒早期抗原)检测EBV在体外能否再感染CNE-2Z细胞,用特异底物法和特异激活剂法分别测定其PKC和TPK活性,MTT法检测CNE-2Z细胞体外增殖能力。结果显示未处理CNE-2Z细胞中PKC活性为膜性>胞核>胞液,TPK为胞核>膜性>胞液。EBV和EBV+TPA再感染CNE-2Z细胞后,抑制细胞增殖,同时胞液PKC和TPK活性升高,膜性和胞核TPK和膜性PKC活性降低。本研究结果提示,EBV可能通过影响不同细胞组分中PKC和TPK活性来调节CNE-2Z鼻咽癌细胞的增殖。     

蛋白激酶C对血管内皮细胞锚蛋白、CD44表达及亚细胞分布的影响

通过外源性底物对[γ-32P]-ATP的摄入量来测定豆蔻酰佛波醇乙酯(phorbol-myristate-acetate,PMA)处理后的人脐静脉内皮细胞(humanumbilicalveinendothelialcells,HUVECs)膜蛋白激酶C(proteinkinaseC,PKC)的活性;利用间接免疫荧光标记和Western印迹方法分析蛋白激酶C活性对锚蛋白及CD44的亚细胞分布及蛋白质表达的影响。结果发现HUVECs的锚蛋白及CD44表达水平趋势与PKC活性变化相吻合;PKC活化导致CD44在细胞膜上呈聚集状,而锚蛋白则移位并聚集于CD44处;PKC抑制剂能抑制PKC活化所带来的上述作用。结果表明PKC活化通过磷酸化作用能上调锚蛋白及CD44表达,并同时导致二者发生一致性运动及共分布。     

普罗托品对大鼠胸主动脉血管平滑肌细胞内游离钙浓度和蛋白激酶C活性的影响

本实验室先前的研究已证实,普罗托品(protopine,Pro)舒张家兔主动脉的作用,可能与其增加血管平滑肌细胞内cAMP和cGMP水平有关.为了深入探讨Pro的扩血管作用机制,实验采用等张收缩记录大鼠离体血管条张力,利用Fura-2/AM负载的大鼠胸主动脉培养细胞直接测定细胞内游离Ca2+浓度([Ca2+]i),并应用同位素γ-32p-ATP催化活性法测定蛋白激酶C(PKC)活性等方法,分别观察了Pro的相关效应.结果表明,Pro(30和100 μmol/L)明显降低去甲肾上腺素(NA)和高钾所致的动脉条收缩幅度,使二者的量效曲线呈非平行右移,最大反应压低;pD2'值分别为3.7±0.25和3.97±0.15;Pro(50和100μmol/L)对静息状态下[Ca2+]i没有任何影响,但对NA和高钾引起的[Ca2+]i升高均有明显抑制作用;Pro(30和100 μmol/L)对未经NA处理血管条的胞浆和胞膜PKC活性均无明显影响;但在NA预处理的血管条,Pro使NA所升高的胞浆内PKC的活性趋于降低,而明显升高胞膜PKC的活性,对PKC的总活性无明显影响.结果提示,在有NA存在的情况下,Pro似能促使PKC从胞浆向细胞膜转移,其扩血管效应似为其降Ca2+作用、升高cAMP和cGMP的作用及其对PKC影响等几方面的综合结果.     

青藤碱对家兔主动脉血管平滑肌细胞内游离钙浓度及蛋白激酶C的影响

观测青藤碱对培养家兔血管平滑肌细胞内游离钙浓度及正常和缺血缺氧刺激下蛋白激酶C（PKC）活性的影响。方法：Fura-2／AM作Ca^2＋指示剂,检测青藤碱对培养家兔主动脉血管平滑肌细胞静息Ca^2＋浓度及去甲肾上腺素,高K^＋,咖啡因刺激作用下的改变,并与钙拮抗剂维拉帕米进行对照研究;复制血管平滑肌细胞缺血缺氧模型,液闪仪测定PKC活性。结果：青藤碱剂量依赖性抑制高K^＋去极化引起[Ca^2＋]i升高,青藤碱10×10^-6mol.L^-1、3×10^-5mol.L^-1、10^-4mol.L^-1,对NE通过受体介导引起的[Ca^2＋]i增高也有明显抑制。但对静息状态下及咖啡因刺激的血管平滑肌细胞[Ca^2＋]i无明显影响。正常时,青藤碱处理后血管平滑肌细胞胞浆、胞膜PKC活性均升高;缺血缺氧状态下,胞浆PKC活性升高,但胞膜PKC活性降低,青藤碱处理后胞浆PKC活性下降,胞膜PKC活性上升。结论：青藤碱可能抑制血管平滑肌细胞电压依赖性钙通道和受体操纵性钙通道,降低细胞内游离钙水平。调节缺血缺氧条件下血管平滑肌细胞PKC活性。     

皮质酮快速激活PC12细胞中p38丝列原激活的蛋白激酶

实验旨在研究糖皮质激素快速、非基因组作用的细胞内信号传导机制。Western分析研究结果表明,皮质酮可快速激活PC12细胞中p38丝列原激活的蛋白激酶（mitogen-activated protein kinase,MAPK）,时间、浓度曲线均为钟形,最大激活为10^-9mol/L和15min。糖皮质激素受体阻断剂RU38486不能阻断此作用,而小牛血清白蛋白耦联的皮质酮也能快速激活p38。受体酪氨酸激酶阻断剂genistein对此作用无影响,表明此快速作用不涉及受体酪氨酸激酶活性。此作用能被蛋白激酶C（protein kinase C,PKC）激动剂PMA模拟,而被PKC阻断剂Goe6976所阻断。结果表明,皮质酮可能通过推测的膜受体以PKC依赖的方式快速激活p38 MAPK。     

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 .     

两种酪氨酸蛋白激酶活性在主动脉平滑肌细胞增殖周期中的动态变化

本文以人工合成的多肽PGAT 为底物鉴定了培养的家兔ASMC 膜性TPK。发现Mg~(2+)和Mn~(2+)对ASMC 胞浆膜性TPK 和核膜性TPK 的激活作用有两点不同:(1)它们对前者的最适激活浓度高于后者;(2)对胞浆膜性TPK,Mn~(2+)最大激活效应大于Mg~(2+)而对核膜性TPK,Mn~(2+)则低于Mg~(2+)。这两种TPK活性在其G_1期的变化特点是:胞浆膜性TPK最高活性出现在G_1晚期(9 h),核膜性TPK最高活性则出现在G_1早期(3 h)。     

组蛋白激活拟南芥CDPK催化CaMBP10的体外磷酸化反应

植物转脂蛋白 (plant lipid transfer proteins, LTPs) 是高等植物中广泛存在的多基因编码的小分子碱性蛋白. 本研究室已经证明白菜和豌豆LTPs可分别被内源胞浆可溶性和膜结合钙依赖性蛋白激酶 (calcium-dependent protein kinase, CDPK) 磷酸化. 为深入研究CDPK对白菜钙调素结合蛋白10 (calmodulin-binding protein-10, CaMBP10) 的磷酸化性质及特征, 本文从拟南芥可溶性蛋白粗提物中检测到1个分子量约为54 kD的CDPK对CaMBP10有磷酸化作用. 研究表明, 组蛋白可增强 CDPK对CaMBP10的磷酸化活性, 促进磷酸化进程. 而且组蛋白和Ca2+对CDPK具有协同调节效应, 二者共同作用时比Ca2+单独作用时, 激酶的活力增强约12倍. 此外, 不同组蛋白对CDPK的激活能力不同, 组蛋白1对该激酶活性的激活能力要比组蛋白3高约8倍.     

麻疯树佛波酯对菜青虫体内几种酶活力的影响

麻疯树佛波酯是麻疯树中最主要的毒性成分。文章研究了取食麻疯树佛波酯后,三龄菜青虫体内的超氧化物歧化酶(SOD)、中肠蛋白酶、Ca2+-ATP酶以及蛋白激酶C(PKC)活力的变化,旨在探讨麻疯树佛波酯对菜青虫的杀虫作用机理。结果表明:取食低浓度的佛波酯刺激菜青虫体内SOD活力增加,而超过16μg·mL-1的处理浓度则会使SOD活力显著下降。麻疯树佛波酯对试虫中肠蛋白酶活力有较强的抑制作用,8μg·mL-1的处理浓度下其比活力显著低于对照组(t测验,P<0.05),随着佛波酯浓度增加,其抑制作用也增大。此外,佛波酯对菜青虫神经细胞膜上Ca2+-ATPase的活性也具有明显的抑制作用。在一定的浓度范围内,菜青虫中肠细胞PKC活力随佛波酯处理浓度升高而升高,64μg·mL-1时PKC比活力最高,达到对照的2.35倍;同时胞膜PKC/胞浆PKC比值升高,伴随着明显的质膜转位。取食麻疯树佛波酯后3h,试虫中肠细胞PKC活力即显著高于对照(t测验,P<0.05),在12h活力达到最大值,为对照组的4.4倍。由于麻疯树佛波酯结构与PKC的激活物二酰基甘油(DAG)类似,因此能显著激活试虫体内的PKC,PKC可能是麻疯树佛波酯发挥杀虫作用的重要靶标。     

Acidic fibroblast growth factor receptor purified from bovine liver is a novel protein tyrosine kinase

Acidic fibroblast growth factor (aFGF) receptor was purified from plasma membranes of bovine liver using Triton X-100 extraction, wheat germ lectin-Sepharose 4B gel affinity chromatography, and DEAE-cellulose anion-exchange chromatography. As previously reported for the aFGF receptor in murine fibroblasts (Huang, S. S., and Huang, J. S. (1986) J. Biol. Chem. 261, 9568-9571), the purified aFGF receptor was also found to be a 135-kDa glycoprotein which showed an intrinsic and ligand-stimulated autophosphorylation activity. The 32P-labeled aFGF receptor was specifically immunoprecipitated by anti-FGF receptor (anti-flg/bek/cek gene product) antiserum. In contrast to other growth factor receptors/protein tyrosine kinases, the protein tyrosine kinase activity (autophosphorylation) of the aFGF receptor was stimulated (approximately 1.5-fold) by low concentrations of Mn2+, Mg2+, and Ca2+ (optimal concentrations of approximately 0.1, approximately 0.1, and 1 microM, respectively) but inhibited by higher concentrations of Mn2+, Mg2+, Ca2+, and pyrophosphate (greater than or equal to 20, greater than or equal to 50, greater than or equal to 10, and greater than or equal to 100 microM, respectively). However, addition of Mn2+ and pyrophosphate at a ratio of 1:1 not only reversed the inhibitory effect but also enhanced the kinase activity about 3-4-fold. The apparent Km of ATP for intrinsic and ligand-stimulated protein kinase activity of the aFGF receptor was estimated to be 25 microM. The preferred exogenous substrates for the protein tyrosine kinase activity of the aFGF receptor were found to be myelin basic protein and histone. Poly-L-arginine, an inhibitor for aFGF binding to the receptor, appeared to stimulate the mitogenesis or cell growth of responsive cells by mimicking aFGF activity.     

The inhibitory effect of BSP-A1/-A2 on protein kinase C and tyrosine protein kinase

Bovine seminal plasma contains a group of similar proteins, namely BSP-A1, BSP-A2, BSP-A3, and BSP-30-kDa (collectively called BSP proteins), and they are secreted by the seminal vesicles. In our study, we purified the BSP-A1/-A2 through affinity chromatography and found for the first time that BSP-A1/-A2 can inhibit the activity of protein kinase C (PKC) and tyrosine protein kinase (TPK). The inhibition was dose dependent. When the PKC and TPK activities are expressed as the logarithm of percentage activity taking the activity in the absence of the BSP-A1/-A2 as 100%, there is a linear relationship between the their activities and the dose of BSP-A1/-A2.     

酪氨酸蛋白激酶和蛋白激酶C对N-乙酰氨基葡萄糖转移酶V的双重调控

在人肝癌细胞７７２１中研究了酪氨酸蛋白激酶（ＴＰＫ）和蛋白激酶Ｃ（ＰＫＣ）的激活剂［分别为表皮生长因子（ＥＧＦ）和佛波酯（ＰＭＡ）］和各种蛋白激酶抑制剂对Ｎ－乙酰氨基葡萄糖转移酶Ｖ（ＧｎＴ－Ｖ）活力的影响,以探讨ＴＰＫ和ＰＫＣ对ＧｎＴ－Ｖ的调节。结果发现,ＥＧＦ或ＰＭＡ处理细胞４８ｈ后,ＧｎＴ－Ｖ的活力明显增高;蛋白激酶的非特异性抑制剂槲皮素和染料木黄酮（ｇｅｎｉｓｔｅｉｎ）在抑制ＴＰＫ和ＰＫＣ的同时,抑制ＧｎＴ－Ｖ的基础活力,并完全阻断ＥＧＦ或ＰＭＡ对ＧｎＴ－Ｖ的增高作用;ＴＰＫ的特异性抑制剂Ｔｙｒｐｈｏｓｔｉｎ－２５和ＰＫＣ的特异性抑制剂Ｄ－鞘氨醇分别应用时,各自只能部分地取消ＥＧＦ或ＰＭＡ对ＧｎＴ－Ｖ的诱导。但当Ｔｙｒｐｈｏｓｔｉｎ－２５和Ｄ－鞘氨醇同时加入培养基中则可完全阻断ＥＧＦ或ＰＭＡ对ＧｎＴ－Ｖ的诱导激活。蛋白质合成抑制剂环己亚胺和蛋白激酶抑制剂作用相仿,不但可抑制ＧｎＴ－Ｖ的基础活力,也可完全消除ＥＧＦ或ＰＭＡ对ＧｎＴ－Ｖ的激活。以上结果提示ＥＧＦ或ＰＭＡ通过蛋白激酶调节ＧｎＴ－Ｖ的酶蛋白合成,并且ＧｎＴ－Ｖ受到膜性ＴＰＫ和ＰＫＣ的双重调节,其中ｍ－ＴＰＫ较ｍ－ＰＫＣ更为重要。     

Intracellular calcium regulates the tyrosine kinase receptor encoded by the MET oncogene.

Previous work (Gandino, L., Di Renzo, M. F., Giordano, S., Bussolino, F., and Comoglio, P.M. (1990) Oncogene 5, 721-725) has shown that the tyrosine kinase activity of the receptor encoded by the MET protooncogene is negatively modulated by protein kinase C (PKC). We now show that an increase of intracellular Ca2+ has a similar inhibitory effect in vivo, via a PKC-independent mechanism. In GTL-16 cells the p145MET kinase is overexpressed and constitutively phosphorylated on tyrosine. A rapid and reversible decrease of p145MET tyrosine phosphorylation was induced by treatment with the calcium ionophores A23187 or ionomycin. Experiments performed with the ionophores in absence of extracellular calcium showed that a rise in cytoplasmic Ca2+ concentration to 450 nM (due to release from intracellular stores) resulted in a similar effect. These Ca2+ concentrations had no effect on p145MET autophosphorylation in an in vitro kinase assay. This suggests that the effect of Ca2+ on p145MET tyrosine phosphorylation is not direct but may be mediated by Ca(2+)-activated proteins(s). Involvement of Ca(2+)-dependent tyrosine phosphatases was ruled out by experiments carried out in presence of Na2VO4. In vivo labeling with [32P]orthophosphate showed that the rise of intracellular Ca2+ induces serine phosphorylation of p145MET on a specific phosphopeptide. This suggests that Ca2+ negatively modulates p145MET kinase through the phosphorylation of a critical serine residue by a Ca(2+)-activated serine kinase distinct from PKC.     

PLC gamma 1, a possible mediator of T cell receptor function

Stimulation of T cell antigen receptor (TCR/CD3) following the recognition of peptide-major histocompatibility antigen complex induces phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolysis. However, the phospholipase C (PLC) enzyme mediating this process has not been identified. We report that PLC gamma 1 protein is expressed in human T cells. It is a phosphoprotein, and the activation of cyclic AMP-dependent protein kinase (PKA) or of protein kinase C (PKC) with forskolin or phorbol ester, respectively, increases the level of phosphorylation. CD3 stimulation of T cells induces tyrosine phosphorylation of PLC gamma 1 and causes 8-10-fold higher yield of PLC activity with anti-phosphotyrosine antibody (APTyr Ab) from activated cells than from non-activated cells. Genistein, an inhibitor of protein tyrosine kinase, decreases this yield of AP-Tyr Ab-bound PLC activity from activated cells and lowers the level of Ca2+ mobilization. Furthermore, phorbol ester and forskolin treatment of cells before CD3 stimulation reduces the level of tyrosine phosphorylation of PLC gamma 1 and the PLC activity associated with APTyr Ab. These results suggest that CD3 stimulation activates PIP2 hydrolysis by inducing tyrosine phosphorylation of PLC gamma 1, which is regulated negatively by PKC and PKA.     

Protein kinase C plays a role in the induction of tyrosine phosphorylation of lymphoid microtubule-associated protein-2 kinase. Evidence for a CD3-associated cascade that includes pp56lck and that is defective in HPB-ALL.

Ligation of the CD3 receptor induces multiple signal transduction events that modify the activation state of the T cell. We have compared two lines that express biologically active CD3 receptors but differ in their biochemical activation pathways during ligation of this receptor. Jurkat cells respond to anti-CD3 with Ca2+ mobilization, PKC activation, induction of protein tyrosine phosphorylation, and activation of newly characterized lymphoid microtubule associated protein-2 kinase (MAP-2K). MAP-2K itself is a 43-kDa phosphoprotein that requires tyrosine phosphorylation for activation. Although ligation of the CD3 receptor in HPB-ALL could stimulate tyrosine phosphorylation of a 59- kDa substrate, there was no associated induction of [Ca2+]i flux, PKC, or MAP-2K activation. A specific PKC agonist, PMA, which bypasses the CD3 receptor, could, however, activate MAP-2K in HPB-ALL cells. This implies that defective stimulation of PKC by the CD3 receptor is responsible for its failure to activate MAP-2K in HPB-ALL. The defect in PKC activation is likely distal to the CD3 receptor as A1F14- failed to activate MAP-2K in HPB-ALL but was effective in Jurkat cells. The stimulatory effect of PMA on MAP-2K activity in HPB-ALL was accompanied by tyrosine phosphorylation of this kinase which implies that PKC may, in some way, regulate tyrosine phosphorylation of MAP-2K. A candidate for this role is pp56lck which underwent posttranslational modification (seen as mobility change on SDS-PAGE) during anti-CD3 and PMA stimulation in Jurkat or PMA treatment in HPB-ALL. There was, in fact, exact coincidence between induction of PKC activity, posttranslational modification of lck and tyrosine phosphorylation/activation of MAP-2K. Lck kinase activity in an immune complex kinase assay was unchanged during PMA treatment. An alternative explanation is that modification of lck may alter its substrate profile. We therefore looked at the previously documented ability of PKC to dissociate lck from the CD4 receptor and found that PMA could reduce the stoichiometry of the lck interaction with CD4 in HPB-ALL and to a lesser extent in Jurkat cells. These results imply the existence of a kinase cascade that is initiated by PKC and, in the course of which, lck and MAP-2K may interact.     

The role of the Ca2+-sensitive tyrosine kinase Pyk2 and Src in thrombin signalling in rat astrocytes

We have recently demonstrated that multiple signalling pathways are involved in thrombin-induced proliferation in rat astrocytes. Thrombin acts by protease-activated receptor-1 (PAR-1) via mitogen-activated protein kinase activity. Signalling includes both Gi/(betagamma subunits)-phosphatidylinositol 3-kinase and a Gq-phospholipase C/Ca2+/protein kinase C (PKC) pathway. In the present study, we investigated the possible protein tyrosine kinases which might be involved in thrombin signalling cascades. We found that, in astrocytes, thrombin can evoke phosphorylation of proline-rich tyrosine kinase (Pyk2) via PAR-1. This process is dependent on the increase in intracellular Ca2+ and PKC activity. Moreover, in response to thrombin stimulation Pyk2 formed a complex with Src tyrosine kinase and adapter protein growth factor receptor-bound protein 2 (Grb2), which could be coprecipitated. Furthermore, both thrombin-induced Pyk2 phosphorylation and extracellular signal-regulated kinase (ERK) 1/2 phosphorylation can be attenuated by Src kinase inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine. From these data we conclude that PAR-1 uses Ca2+- and PKC-dependent Pyk2 to activate Src, thereby leading to ERK1/2 activation, which predominantly recruits Grb2 in rat astrocytes.     

Kinases,intracellular calcium,and apolipophorin-III influence the adhesion of larval hemocytes of the lepidopterous insect,Galleria mellonella

Based on the results from the use of selective inhibitors and activators, active protein kinase A, protein tyrosine kinase, and protein kinase C (PKC) isoforms decreased the adhesion of larval Galleria mellonella hemocytes to glass slides. The protein kinase A inhibitor at all concentrations increased granular cell adhesion only whereas protein tyrosine kinase elevated both granular and plasmatocyte attachment at the lowest concentration. Active, Ca(2+)- and lipid-dependent PKC isoforms limited plasmatocyte and granular cell adhesion whereas PKC that was inhibited by selected compounds (with differed modes of PKC inhibition) enhanced hemocyte attachment. The granular cells were more sensitive to the PKC inhibitors than were plasmatocytes. Phospholipase C and its diacylglyceride product were necessary to reduce hemocyte adhesion and maintain PKC activity. Extracellular Ca(2+), possibly transported through L-channels, was required for plasmatocyte attachment. In contrast, lowering the levels of cytosolic Ca(2+) was associated with decreased PKC activity and was required for hemocyte adhesion.     

ANG II-stimulated DNA synthesis is mediated by ANG II receptor-dependent Ca(2+)/PKC as well as EGF receptor-dependent PI3K/Akt/mTOR/p70S6K1 signal pathways in mouse embryonic stem cells

Effect of angiotensin II (ANG II) on mouse embryonic stem (ES) cell proliferation was examined. ANG II increased [(3)H] thymidine incorporation in a time- (>4 h) and dose- (>10(-9) M) dependent manner. The ANG II-induced increase in [(3)H] thymidine incorporation was blocked by inhibition of ANG II type 1 (AT(1)) receptor but not by ANG II type 2 (AT(2)) receptor, and AT(1) receptor was expressed. ANG II increased inositol phosphates formation and [Ca(2+)](i), and translocated PKC alpha, delta, and zeta to the membrane fraction. Consequently, the inhibition of PLC/PKC suppressed ANG II-induced increase in [(3)H] thymidine incorporation. The inhibition of EGF receptor kinase or tyrosine kinase prevented ANG II-induced increase in [(3)H] thymidine incorporation. ANG II phosphorylated EGF receptor and increased Akt, mTOR, and p70S6K1 phosphorylation blocked by AG 1478 (EGF receptor kinase blocker). ANG II-induced increase in [(3)H] thymidine incorporation was blocked by the inhibition of p44/42 MAPKs but not by p38 MAPK inhibition. Indeed, ANG II phosphorylated p44/42 MAPKs, which was prevented by the inhibition of the PKC and AT(1) receptor. ANG II increased c-fos, c-jun, and c-myc levels. ANG II also increased the protein levels of cyclin D1, cyclin E, cyclin-dependent kinase (CDK) 2, and CDK4 but decreased the p21(cip1/waf1) and p27(kip1), CDK inhibitory proteins. These proteins were blocked by the inhibition of AT(1) receptor, PLC/PKC, p44/42 MAPKs, EGF receptor, or tyrosine kinase. In conclusion, ANG II-stimulated DNA synthesis is mediated by ANG II receptor-dependent Ca(2+)/PKC and EGF receptor-dependent PI3K/Akt/mTOR/p70S6K1 signal pathways in mouse ES cells.