Miettinen S, Roivainen R, Keinänen R, Hökfelt T, Koistinaho J
A. I. Virtanen Institute, University of Kuopio, Finland.
J Neurosci. 1996 Oct 1;16(19):6236-45. doi: 10.1523/JNEUROSCI.16-19-06236.1996.
Protein kinase C (PKC) consists of a family of closely related Ca2+/phospholipid-dependent phosphotransferase isozymes, most of which are present in the brain and are differentially activated by second messengers. Calcium-dependent PKC activity may cause neuronal degeneration after ischemic insult. PKC is also involved in trophic-factor signaling, indicating that activity of some PKC subspecies may be beneficial to the injured brain. Therefore, we screened long-term changes in the expression of multiple PKC subspecies after focal brain ischemia. Middle cerebral artery occlusion was produced by using an intraluminal suture for 30 min of 90 min. In in situ hybridization experiments, mRNA levels of PKC alpha, -beta, -gamma, -delta, -epsilon, and -zeta were decreased in the infarct core 4 hr after ischemia and were lost completely 12 hr after ischemia. In areas surrounding the core, PKC delta mRNA was specifically induced 4, 12, and 24 hr after ischemia in the cortex. At 3 and 7 d, the core and a rim around it showed increased mRNA levels of PKC delta. No other subspecies were induced. At 2 d, immunoblotting demonstrated increased levels of PKC delta protein in the perifocal tissue, and immunocytochemistry revealed an increased number of PKC delta-positive neurons in the perifocal cortex. In the core, PKC delta-positive macrophages and endothelial cells were seen. Pretreatment with MK-801, an NMDA antagonist, inhibited cortical PKC delta mRNA induction. The data show that focal brain ischemia induces PKC delta mRNA and protein but not other PKC subspecies through the activation of NMDA receptors and that the upregulation lasts for several days in neurons of the perifocal zone.
蛋白激酶C（PKC）由一族密切相关的钙/磷脂依赖性磷酸转移酶同工酶组成，其中大多数存在于大脑中，并被第二信使差异性激活。缺血性损伤后，钙依赖性PKC活性可能导致神经元变性。PKC也参与营养因子信号传导，这表明某些PKC亚型的活性可能对受损大脑有益。因此，我们筛选了局灶性脑缺血后多种PKC亚型表达的长期变化。采用腔内缝合线闭塞大脑中动脉30分钟或90分钟。在原位杂交实验中，缺血4小时后梗死核心区PKCα、β、γ、δ、ε和ζ的mRNA水平降低，缺血12小时后完全消失。在核心区周围，缺血后4、12和24小时，皮质中PKCδmRNA被特异性诱导。在3天和7天时，核心区及其周围边缘显示PKCδ的mRNA水平升高。没有诱导出其他亚型。在2天时，免疫印迹显示病灶周围组织中PKCδ蛋白水平升高，免疫细胞化学显示病灶周围皮质中PKCδ阳性神经元数量增加。在核心区，可见PKCδ阳性巨噬细胞和内皮细胞。用NMDA拮抗剂MK-801预处理可抑制皮质PKCδmRNA的诱导。数据表明，局灶性脑缺血通过激活NMDA受体诱导PKCδmRNA和蛋白，但不诱导其他PKC亚型，并且上调在病灶周围区的神经元中持续数天。
