Effects of Depolarization and NMDA Antagonists on the Survival of Cerebellar Granule Cells: A Pivotal Role for Protein Kinase C Isoforms
Effects of Depolarization and NMDA Antagonists on the Survival of Cerebellar Granule Cells: A Pivotal Role for Protein Kinase C Isoforms
复制标题
去极化和 NMDA 拮抗剂对小脑颗粒细胞存活的影响：蛋白激酶 C 异构体的关键作用
作者：
Wan;Ching;D. Chuang
摘要：在高K+(25 mM；K25)培养液中培养的小脑颗粒细胞(CGCs)在体外呈渐进性分化。在体外培养3-4天(DIV)时分化明显，8天后分化成熟。长期培养CGCs(>；13DIV)会触发自发的细胞死亡过程。然而，如果在正常的生理K+浓度(5 mM；K5)中培养，相当大比例的细胞在培养的第一周结束时死亡。为了探讨蛋白激酶C(PKC)在CGCs发育中的作用，我们测定了蛋白激酶C的活性和蛋白水平。随着K25CGC培养的进行，PKC活性呈时间依赖性增加3.2倍，在8DIV时达到稳定状态。使用蛋白激酶C亚型特异性抗体的Western印迹分析显示，蛋白激酶Cα，γ，μ，λ和ι水平从2DIV增加到8DIV。在4DIV时，PKCε和βII分别略有增加或减少，而βI则无明显变化。未检测到δ，θ，η和ζ亚型。14种DIV与10种DIV相比，PKCι和ε的免疫反应活性降低，PKCα，βI、βII、γ和λ的免疫反应活性没有变化，而PKCμ的免疫反应活性仍然增加。在K5细胞中，2-4DIV时各PKC亚型的免疫反应性与K25细胞相似，但未观察到明显的分化特征。随着低K+培养8DIV时细胞死亡的出现，PKC、α，μ，λ和ι的表达水平显著降低，而其他蛋白表达水平无明显变化。NMDA受体拮抗剂MK-801和2-氨基-5-磷酸戊酸可显著抑制AGE诱导的CGCs的凋亡，并使细胞存活。在10d和14d，MK-801的细胞保护作用伴随着PKCγ，λ，ι和μ水平的升高。此外，与单独培养的K25相比，PKCε水平在14DIV时升高，但在早期下降，而PKCα，βI和βII水平不变。综上所述，PKC亚型的诱导和上调可能在去极化和MK-801维持CGC存活中发挥重要作用。
Abstract: Primary cultures of cerebellar granule cells (CGCs) grown in high‐K+ (25 mM; K25) medium progressively differentiate in vitro. Differentiation is noticeable after 3–4 days in vitro (DIV) and reach a mature stage after 8 DIV. Longer cultivation of CGCs (>13 DIV) triggers the processes of spontaneous cell death. However, if cultured in normal physiological K+ concentration (5 mM; K5), a significant proportion of the cells dies by the end of the first week in culture. To address the role of protein kinase C (PKC) in the development of CGCs, we measured the kinase activity as well as the protein level of the kinase isoforms. As the K25 CGC culture proceeded, the PKC activity time‐dependently increased by 3.2‐fold, reaching a steady state at 8 DIV. Western blot analysis using PKC isoform‐specific antibodies revealed an increase in levels of PKC α, γ, μ, λ, and ι from 2 to 8 DIV. A slight increase or decrease at 4 DIV was observed for PKC ε and βII, respectively, whereas no significant change was observed for βI. The isoforms of δ, θ, η, and ζ were not detected. Comparing the 14 DIV cultures with the 10 DIV cultures, the immunoreactivities of PKC ι and ε were decreased, those of PKC α, βI, βII, γ, and λ were unchanged, whereas that of PKC μ was still increased. In K5 cultures, the immunoreactivity of each PKC isoform at 2–4 DIV was similar to that observed in K25 cells, although no remarkable differentiation features were observed. Coordinated with the appearance of cell death at 8 DIV in low‐K+ cultures, levels of PKC α, μ, λ, and ι, but not the others, were markedly decreased. The NMDA receptor antagonists MK‐801 and 2‐amino‐5‐phosphopentanoic acid markedly prevented the age‐induced apoptosis of CGCs, and the cells survived >18 DIV under these conditions. The cytoprotective effect of MK‐801 was concomitant with the increases in levels of PKC γ, λ, ι, and μ at 10 and 14 DIV. In addition, the PKC ε level was increased at 14 DIV but decreased at early stages, whereas PKC α, βI, and βII levels were unchanged, as compared with K25 culture alone. Taken together, induction and up‐regulation of PKC isoforms may play an important role in the maintenance of CGC survival by depolarization and MK‐801.
神经生长因子活性所需的苔藓抑素敏感蛋白激酶 C。
影响因子：
2.9
作者：
Singh,KR;Taylor,LK;Campbell,XZ;Fields,AP;Neet,KE
通讯作者：
Neet,KE
神经生长因子诱导的神经突发生过程中 PC12 细胞中蛋白激酶 C-δ 的选择性易位。
影响因子：
3.3
作者：
O'Driscoll,KR;Teng,KK;Fabbro,D;Greene,LA;Weinstein,IB
通讯作者：
Weinstein,IB
DOI：
10.1073/pnas.87.4.1620
发表时间：
1990-02-01
影响因子：
11.1
作者：
BECKMAN, JS;BECKMAN, TW;FREEMAN, BA
通讯作者：
FREEMAN, BA