甾体激素对神经母细胞株SK-N-SH细胞摄取甘氨酸的快速作用

本研究观察了ＳＫ－Ｎ－ＳＨ细胞摄取甘氨酸的一般情况,并进一步研究了甾体激素对ＳＨ－Ｎ－ＳＨ细胞摄取甘氨酸的快速作用。结果显示：ＳＫ－Ｎ－ＳＨ细胞高亲和力的甘到摄取是Ｎａ＾＋和Ｃｌ＾－依赖性的。皮质酮、孕酮和地塞米松对这种摄取有快速促进作用,雌二醇和脱氧皮质酮无明显作用,表明甾体激素快速作用有特异性。皮质酮作用在１０＾－９￣１０＾－６ｍｏｌ／Ｌ范围内呈浓度依赖性。此质酮快速作用不受蛋白质合成抑制影响     

LA－90细胞在温度转化过程中蛋白质酪氨酸磷酸化作用研究

ＬＡ－９０细胞在温度转化过程中蛋白质酪氨酸磷酸化作用研究夏英,高漫,颜卉君,吴国利（北京师范大学生物系生物化学及分子生物学研究室,１００８７５）关键词酪氨酸蛋白激酶;磷酸酪氨酸蛋白磷酸酶;细胞转化ｉｓ－ＲＳＶＬＡ－９０细胞是ＲＳＶ转染的小鼠３Ｔ３细胞...     

AVP（4—8）增强CK—N—XH细胞内PKC和MAPK的活性

ＡＶＰ（４￣８）是精氨酸加压素（ＡＶＰ）在脑内的天然酶解产物,具有增强学习记忆的功能。为了进一步阐明其作用的分子机制,以ＳＫ－Ｎ－ＳＨ成神经瘤细胞（ＳＫ细胞）为模型进行研究。放射性配基结合实验表明,在ＳＫ细胞上存在ＡＶＰ（４￣８）的特异性结合位点。ＡＶＰ（４￣８）可以刺激ＳＫ细胞中蛋白激酶Ｃ（ＰＫＣ）和促细胞分裂原活化的蛋白激酶（ＭＡＰＫ）尖性的升高,并可以被ＡＶＰ（４￣８）的受体拮抗剂ＺＤＣ（Ｃ     

星形胶质细胞和神经元之间谷氨酸-谷氨酰胺的代谢偶联

谷氨酸-谷氨酰胺循环是星形胶质细胞和神经元代谢偶联最重要的途径之一。在中枢神经系统中葡萄糖经糖酵解和三羧酸循环,合成三羧酸循环的中间产物。神经元因缺乏丙酮酸羧化酶,不能由葡萄糖直接合成谷氨酸,而必须依赖于星形胶质细胞的三羧酸循环来产生作为谷氨酸前体的三羧酸循环中间代谢产物。星形胶质细胞的谷氨酸载体从突触间隙摄取谷氨酸,在星形胶质细胞中转变成谷氨酰胺并释放到细胞外,然后重新被神经元摄取,转变成谷氨酸进入新一轮的循环。本文介绍了该循环,以及星形胶质细胞谷氨酸载体的功能、特性及调控。     

丝裂素活化蛋白激酶参与内皮素刺激的兔主动脉平滑肌细胞增生

内皮素（ｅｎｄｏｔｈｅｌｉｎ,ＥＴ）是已知的体内活性最强的缩血管物质,其缩血管作用由Ｇ蛋白偶联受体所介导。但ＥＴ强大的促血管平滑肌细胞（ＶＳＭＣ）增生效应的机理尚未完全阐明。本研究选用培养的兔胸主动脉ＶＳＭＣ,探讨丝裂素活化蛋白激酶（ＭＡＰＫ）在ＥＴ促细胞增生中的作用。结果表明：ＥＴ－１呈时间和浓度依赖性地促进细胞摄取￣３Ｈ－ＴｄＲ和激活ＭＡＰＫ,此作用可被蛋白激酶Ｃ（ｐｒｏｔｅｉｎｋｉｎａｓｅＣ,ＰＫＣ）抑制剂Ｓｔａｕｒｏｓｐｏｒｉｎｅ（ＳＴＰ）,Ｈ－７和ＥＴ＿Ａ受体拮抗剂ＢＱ１２３所抑制,但不被酪氨酸激酶抑制剂ＨｅｒｂｉｍｙｃｉｎＡ（Ｈｅｒｂ）所抑制,用ＰＫＣ激动剂ＰＭＡ（Ｐｈｏｒｂｏｌｍｙｒｉｓｔａｔｅａｃｅｔａｔｅ）预处理ＶＳＭＣ,使其ＰＫＣ活性下调,可显著减弱ＥＴ－１对ＭＡＰＫ的激活能力。本结果提示：（１）ＭＡＰＫ参与ＥＴ－１所致的ＶＳＭＣ增生;（２）ＥＴ－１促细胞增生与激活ＭＡＰＫ的作用是由ＥＴ＿Ａ受体和ＰＫＣ介导的。     

高亲和力谷氨酸转运体

高亲和力谷氨酸转运体主要位于神经元和胶质细胞的细胞膜上,能逆浓度梯度从胞外向胞内摄取谷氨酸,中止谷氨酸能传递,使胞外谷氨酸浓度保持在较低水平,以保护神经元不受谷氨酸的毒性影响。近年来,随着高亲和力谷氨酸转运体的克隆,有关研究迅速发展。本文从高亲和力谷氨酸转运体的克隆、分子结构特征、表达分布、生理功能、结构－功能关系等方面对近年的进展加以综述。     

蛋白激酶C对N－乙酰氨基葡萄糖转移酶Ⅲ的调节

人肝癌细胞株７７２１细胞的Ｎ－乙酰氨基葡萄糖转移酶Ⅲ（ＧｎＴⅢ）活性受Ｓｅｒ／Ｔｈｒ蛋白激酶的两种抑制剂ｑｕｅｒｃｅｔｉｎ和三氟吡嗪（ＴＦＰ）．蛋白激酶Ｃ（ＰＫＣ）的两种特异性抑制剂Ｄ－鞘氨醇和ｓｔａｕｒｏｓｐｏｒｉｎｅ的抑制。用ＰＭＡ处理细胞舌,ＧｎＴⅢ活力随膜性ＰＫＣ（ｍ－ＰＫＣ）活力而平行变化,但与胞液ＰＫＣ活力的变化无关。Ｑｕｅｒｃｅｔｉｎ、Ｄ－鞘氨醇和ｓｔａｕｒｏｓｐｏｒｉｎｅ还能够阻断ＰＭＡ对ＧｎＴⅢ的激活。Ｑｕｅｒｃｅｔｉｎ、ｓｔａｕｒｏｓｐｏｒｉｎｅ对ｍ－ＰＫＣ和ＧｎＴⅢ的抑制作用基本上与它们的应用浓度成正比关系。当人及大鼠肾脏的粗ＧｎＴ制剂分别用碱性磷酸酶切除磷酸基后,ＧＤＴⅢ的活力明显下降。这些结果表明ｍ－ＰＫＣ可能通过蛋白质的Ｓｅｒ／Ｔｈｒ残基上磷酸化和去磷酸化作用直接或间接地调节ＧｎＴⅢ。     

一氧化氮抑制内皮素促血管平滑肌细胞增殖作用的信号转导途径

培养的家兔胸主动脉血管平滑肌细胞（ＶＳＭＣ）分别以内皮素（ＥＴ－１）、一氧化氮（ＮＯ）前体Ｌ－Ａｒｇ和ＮＯ供体ＳＩＮ－１刺激,或用ＥＴ－１＋Ｌ－Ａｒｇ、ＥＴ－１＋ＳＩＮ－１联合刺激,测ＶＳＭＣ＾３Ｈ－ＴｄＲ掺入、丝裂素活化蛋白激酶（ＭＡＰＫ）活性及蛋白激酶Ｃ（ＰＫＣ）活性的改变,以研究ＮＯ抑制ＥＴ－１促ＶＳＭＣ增殖作用的信号转导途径。结果表明：（１）ＥＴ－１ １０＾－８ｍｏｌ／Ｌ单独刺激,＾３Ｈ－     

Ephrin-A3逆向通路对海马星形胶质细胞谷氨酸摄取能力的影响

目的观察EphA4介导的ephrin-A3逆向通路激活对星形胶质细胞谷氨酸摄取能力的影响。方法采用原代培养的大鼠海马星形胶质细胞,使用免疫荧光双标法定位ephrin-A3在海马星形胶质细胞上的表达,Western blot法观察糖氧剥夺(oxygen-glucose deprivation,OGD)后星形胶质细胞ephrin-A3表达水平的变化,随后实验分为三组:空白对照组(不含星形胶质细胞),药物对照组(加入IgG-Fc)和EphA4组(加入ephrin-A3逆向通路激动剂预聚集化的EphA4-Fc),分别在正常及缺血条件下的特定时间点以谷氨酸浓度测定试剂盒检测不同干预组星形胶质细胞谷氨酸摄取能力的变化。结果 ephrin-A3高表达于海马星形胶质细胞,并在缺血后出现蛋白表达水平一过性上调。与对照组相比,EphA4干预组星形胶质细胞谷氨酸摄取能力较对照组明显下降。结论 Ephrin-A3高表达于海马星形胶质细胞并参与调节星形胶质细胞谷氨酸摄取能力。     

类卟啉稀土配合物对于小鼠腹水肝癌细胞光敏损伤的研究

研究了类卟啉稀土配合物（以下简称ＰＬＭ－Ｇｄ－Ａ）对小鼠腹水肝癌细胞（ＡＨ）的光敏作用及ＡＨ细胞对ＰＬＭ－Ｇｄ－Ａ的摄取表明：ＰＬＭ－Ｇｄ－Ａ被ＡＨ细胞摄取的速度快,用ＭＴＴ方法测定了细胞光敏存活曲线,杀伤细胞的能力与光照时间和光照强度以及ＰＬＭ－Ｇｄ的浓度密切相关,用ＦＡＤＵ方法和电镜观察结果证明：该光敏损伤细胞的靶主要是在细胞核;对于不同稀土离子配合物光敏能力比较表明：Ｇｄ＞Ｅｕ＞Ｓｍ;造成细     

佛波酯引起蛋白激酶C下降调节的专一性

探讨了佛波酯（ＰＭＡ）对蛋白激酶的下降调节是否有激酶专一性及亚型专一性．用组蛋白Ｈ１作为蛋白激酶Ｃ（ＰＫＣ）和蛋白激酶Ａ（ＰＫＡ）的受体底物,加入ＰＫＣ和ＰＫＡ的特异性激活剂区分ＰＫＣ和ＰＫＡ,用聚谷酪（４１）为酪氨酸蛋白激酶（ＴＰＫ）的专一性受体底物,以３２Ｐ－ＡＴＰ为３２Ｐ共同供体底物测定三种蛋白激酶的活力,并用免疫组化法测定ＰＫＣ亚型．结果发现ＰＭＡ对人７７２１肝癌细胞只引起ＰＫＣ而不引起ＰＫＡ和ＴＰＫ的下降调节,ＰＫＣ的非特异性抑制剂槲皮素和特异性抑制剂Ｄ－鞘氨醇能大部分取消ＰＭＡ对ＰＫＣ的下降调节,但ＴＰＫ抑制剂ｇｅｎｅｓｔｅｉｎ则没有阻断下降调节的作用．用ＨＬ－６０细胞还证明ＰＭＡ只对含量丰富的ＰＫＣα和ＰＫＣβⅡ亚型而不对含量很少的ＰＫＣβⅠ亚型发生下降调节．上述结果说明ＰＭＡ对蛋白激酶的下降调节有激酶和亚型专一性．     

Modulation of succinate transport in Hep G2 cell line by PKC

The cellular uptake of the tricarboxylic acid cycle (TCA) intermediates is very important for cellular metabolism. However, the transport pathways for these intermediates in liver cells are not well characterized. We have examined the transport of succinate and citrate in the human hepatoma cell line Hep G2 and found that it exhibited a higher rate of succinate compared to citrate transport, which was sodium dependent. Comparison of the transport properties of Hep G2 to that of human retinal pigment epithelial (HRPE) cells transfected with human sodium dicarboxylate transporters, hNaDC-1, hNaDC-3, and hNaCT indicated that Hep G2 cells express a combination of hNaDC-3 and hNaCT. Short period activation of protein kinase C (PKC) by phorbol 12-myristate, 13-acetate (PMA) and α-adrenergic receptor agonist, phenylephrine (PE), downregulated sodium-dependent succinate transport presumably via hNaDC-3. The inhibition by PMA was partially prevented by cytochalasin D, suggesting that PKC reduces the hNaDC-3 activity, at least in part, by increased endocytosis. In contrast, activation of PKA by both forskolin and epidermal growth factor (EGF) had no effect on succinate transport. Our results suggest that Hep G2 cells provide a useful model for studies of di- and tricarboxylate regulation of human liver.     

Modulation of succinate transport in Hep G2 cell line by PKC

The cellular uptake of the tricarboxylic acid cycle (TCA) intermediates is very important for cellular metabolism. However, the transport pathways for these intermediates in liver cells are not well characterized. We have examined the transport of succinate and citrate in the human hepatoma cell line Hep G2 and found that it exhibited a higher rate of succinate compared to citrate transport, which was sodium dependent. Comparison of the transport properties of Hep G2 to that of human retinal pigment epithelial (HRPE) cells transfected with human sodium dicarboxylate transporters, hNaDC-1, hNaDC-3, and hNaCT indicated that Hep G2 cells express a combination of hNaDC-3 and hNaCT. Short period activation of protein kinase C (PKC) by phorbol 12-myristate, 13-acetate (PMA) and alpha-adrenergic receptor agonist, phenylephrine (PE), downregulated sodium-dependent succinate transport presumably via hNaDC-3. The inhibition by PMA was partially prevented by cytochalasin D, suggesting that PKC reduces the hNaDC-3 activity, at least in part, by increased endocytosis. In contrast, activation of PKA by both forskolin and epidermal growth factor (EGF) had no effect on succinate transport. Our results suggest that Hep G2 cells provide a useful model for studies of di- and tricarboxylate regulation of human liver.     

Differential regulation by protein kinases of activity and cell surface expression of glutamate transporters in neuron-enriched cultures

This study described the involvement of short-term PKA, PKC or PI3K phosphorylation-mediated processes in the regulation of activity and trafficking of the excitatory amino acid transporters EAAC1, GLAST and GLT-1 endogenously expressed in neuron-enriched cultures. Glutamate uptake was dose-dependently decreased by inhibitors of protein kinase A (PKA), [N-[2-(p-bromocinnamylamino)-ethyl]-5-(isoquinolinesulfonamide)] (H89) or phosphatidylinositol 3-kinase (PI3K) (wortmannin), but not altered after protein kinase C (PKC) inhibition (staurosporine) or activation phorbol-12-myristate-13-acetate (PMA). Biotinylation and immunoblotting results (% of controls) showed that EAAC1 membrane expression was significantly decreased by H89 (71.9+/-4.7%) and wortmannin (63.3+/-20.0%) and increased by PMA (137.7+/-15.5%). H89 and PMA induced a significant decrease of the cell surface fraction of GLAST (54.0+/-34.1% and 73.3+/-14.3%, respectively) whereas wortmannin significantly increased this fraction (119.8+/-9.3%). After treatment with H89, the GLT-1 membrane level showed a two-fold increase (179.4+/-19.7%). Conversely, PMA and wortmannin induced a significant decrease of the cell surface expression of GLT-1 (49.0+/-15.4% and 40.7+/-33.7%, respectively). Confocal microscopy revealed a wortmannin-induced clustering of EAAC1 in the intracellular compartment. These data suggest that trafficking of glutamate transporters can be differentially regulated by PKA-, PKC- and PI3K-dependent signaling pathways and could therefore control total glutamate uptake activity. These processes may represent rapid adaptive responses to changes in the cellular environment, which significantly contribute to regulation of EAA transmission and further prevent possible excitotoxic events.     

Protein Kinase C Modulates Calcium Sensitivity of Nitric Oxide Synthase in Cerebellar Slices

Abstract: The possible modulation of nitric oxide (NO) synthase (NOS) activity by protein kinase C (PKC) was investigated. Incubation of rat cerebellar slices with the specific metabotropic glutamate receptor agonist, (±)-1-aminocyclopentane- trans -1,3-dicarboxylate ( trans -ACPD) increased cyclic GMP concentration two-fold. The increase was dose-dependently blocked by the protein kinase inhibitors staurosporine and calphostin C. Phorbol 12-myristate 13-acetate (PMA), a PKC activator, increased cyclic GMP concentration without glutamate receptor activation. The cyclic GMP increases induced by PMA and trans -ACPD were independent of extracellular calcium blocked by N ^ω-nitro- l -arginine, a specific NOS inhibitor, and were not additive. Measurement of citrulline formation in cerebellar slices confirmed that NOS was activated by trans -ACPD and the activation was blocked by calphostin C. These results suggest that metabotropic glutamate receptor activates NOS through PKC. The calcium dependency of NOS activation was assessed in slices incubated with PMA and okadaic acid. NOS in both PMA-treated and untreated slices had similar activities at 100 n M free calcium, whereas at 25–70 n M free calcium, NOS in PMA-treated slices was more active than that in untreated slices. These results suggest that PKC regulates NO release in resting neurons by modulating the sensitivity of NOS at low calcium concentrations.     

The role of protein kinase C-delta in PTH stimulation of IGF-binding protein-5 mRNA in UMR-106-01 cells

We have investigated the role of protein kinase C (PKC) signal transduction pathways in parathyroid hormone (PTH) regulation of insulin-like growth factor-binding protein-5 (IGFBP-5) gene expression in the rat osteoblast-like cell line UMR-106-01. Involvement of the PKC pathway was determined by the findings that bisindolylmaleimide I inhibited 40% of the PTH effect, and 1 microM bovine PTH-(3-34) stimulated a 10-fold induction of IGFBP-5 mRNA. PTH-(1-34) and PTH-(3-34) (100 nM) both stimulated PKC-delta translocation from the membrane to the nuclear fraction. Rottlerin, a PKC-delta-specific inhibitor, and a dominant negative mutant of PKC-delta were both able to significantly inhibit PTH-(1-34) and PTH-(3-34) induction of IGFBP-5 mRNA, suggesting a stimulatory role for PKC-delta in the effects of PTH. Phorbol 12-myristate 13-acetate (PMA) stimulated PKC-alpha translocation from the cytosol to the membrane and inhibited approximately 50% of the PTH-(1-34), forskolin, and 8-bromoadenosine 3',5'-cyclic monophosphate-stimulated IGFBP-5 mRNA levels, suggesting that PKC-alpha negatively regulates protein kinase A (PKA)-mediated induction of IGFBP-5 mRNA. These results suggest that the induction of IGFBP-5 by PTH is both PKA and PKC dependent and PKC-delta is the primary mediator of the effects of PTH via the PKC pathway.     

Resistance to induced apoptosis in the human neuroblastoma cell line SK-N-SH in relation to neuronal differentiation. Role of Bcl-2 protein family.

Much evidence suggests that apoptosis plays a crucial role in cell population homeostasis that depends on the expression of various genes implicated in the control of cell life and death. The sensitivity of human neuroblastoma cells SK-N-SH to undergo apoptosis induced by thapsigargin was examined. SK-N-SH were previously differentiated into neuronal cells by treatments with retinoic acid (RA), 4 beta-phorbol 12-myristate 13-acetate (PMA) which increases protein kinase C (PKC) activity, and staurosporine which decreases PKC activity. Neuronal differentiation was evaluated by gamma-enolase, microtubule associated protein 2 (MAP2) and synaptophysin immunocytochemistry. The sensitivity of the cells to thapsigargin-induced apoptosis was evaluated by cell viability and nuclear fragmentation (Hoechst 33258) and compared with pro-(Bcl-2, Bcl-x(L)) and anti-apoptotic (Bax, Bak) protein expression of the Bcl-2 family. Cells treated with RA and PMA were more resistant to apoptosis than controls. Conversely, the cells treated with staurosporine were more susceptible to apoptosis. In parallel with morphological modifications, the expression of inhibitors and activators of apoptosis was directly dependent upon the differentiating agent used. Bcl-2 expression was strongly increased by PMA and drastically decreased by staurosporine as was Bcl-x(L) expression. Bax and Bak expression were not significantly modified. These results demonstrate that drugs that modulate PKC activity may induce a modification of Bcl-2 expression as well as resistance to the apoptotic process. Furthermore, the expression of Bcl-2 was reduced by toxin B from Clostridium difficile and, to a lesser extent, by wortmannin suggesting a role of small G-protein RhoA and PtdIns3 kinase in the control of Bcl-2 expression. Our data demonstrate a relationship between the continuous activation of PKC, the expression of Bcl-2 protein family and the resistance of differentiated SK-N-SH to apoptosis.