| Previous studies indicate that vascular endothelial growth factor (VEGF) has direct neurotrophic action and prevents ischemic neuronal injury; it also can inhibit the outward delayed-rectifier potassium currents (/K) in the neurons. Over-activation of delayed-rectifier potassium channel, which leads to an augmentation of IK, is associated with the delayed neuronal death in ischemic/repefusion injury. So the inhibition of /K is maybe one of the mechanisms of VEGF's neuroprotection effects. It has been demonstrated that Kvl.2, an isoform of delayed-rectifier potassium channel, is up-regulated in the ischemic neurons. Besides, the activity of Kvl.2 is tyrosine phosphorylation dependent, phosphorylation of the tyrosine residue in the amino terminus of the channel by tyrosine kinases suppresses the channel current. Therefore, we hypothesize that VEGF could enhance the phosphorylation of Kvl.2, which results in an inhibition of /K. To make clear the hypothesis, a middle cerebral artery occlusion (MCAo) and hypoxia and glucose-deprivation (HGD) ischemic models were used in in vivo and in vitro study, respectively. We first studied the cellular localization of Kvl.2 and the co-localization of Kvl.2 protein and VEGF receptors Flk-1 and Flt-1, observed the effect of MCAo on Kvl.2 expression and phosphrylation in the rat brain in vivo, then investigated the effect of VEGF on ischemia/hypoxia cell damage and tyrosine phosphorylation of Kvl.2 in SH-SY5Y cells. Finally, in order to further elucidate the relationship between VEGF's neuroprotection and its regulation on Kvl.2 phosphorylation, we used a specific antisense oligodeoxynucleotide (ODN) to knockdown the expression of endogenous VEGF to observe its role in ischemia/hypoxia cell damage and regulation of Kvl.2 phosphorylation.The results were summarized as follows:1. The cellular localization of Kvl.2 and the co-localization of Kvl.2 and VEGF receptors in the rat brainIn rat cerebral cortex, triple-fluorescence labeling for Kvl.2, NSE, and GFAP showed that Kvl.2 immunopositive labeling was predominantly seen on themembrane of the cells co-stained with NSE, a specific neuronal marker. Only few Kvl.2 positive labeling cells were co-stained with GFAP, an astrocyte glial cell marker.In rat cerebral cortex, double-fluorescence labeling for Kvl.2 and Flk-l/Flt-1 showed that there were many cells expressing Flk-1, and most of them were co-labeled with Kvl.2. Little cell expressed Flt-1 protein, and no double-labeled Kvl.2 and Flt-1 positive cell was found. In CA1 area of rat hippocampus, triple-fluorescence labeling for Kvl.2, Flk-1, and Flt-1 showed that the distribution of Kvl.2 and Flk-1 was identical, they mainly distributed on the membrane and the neuritis of the cells exhibiting a typical morphology of pyramidal cell of CA1 area. The distribution of Flt-1 in this area was similar with that in cerebral cortex, and also has no co-localization with Kvl.2. The result indicated that Kvl.2 co-localized with Flk-1 receptor.2. Ischemia induced expression and tyrosine residue phosphorylation of Kvl.2.In in vivo study. In the rat brain following MCAo for 30 min with 3 days reperfusion, immunohistochemical staining showing that the Kvl.2 immuno-like positive staining was highly enhanced in the ipsilateral hemisphere, especially in the cerebral cortex. Immunoprecipitation combining Western blot analysis showing that the phosphorylation level of Kvl.2 was increased in the ischemic injured cerebral cortex by factors of 3.32, 2.56 and 1.93 at 0 hr, 6 hr and 1 d, respectively, and returned to the control level at 3 days later.In in vitro study: In the SH-SY5Y cells, the Kvl.2 protein was at low level without HGD treatment. However, after HGD treatment, the protein level of Kvl.2 began to increase after 1 hr, reached a maximum after 1 5 hr, and reduced back after 24 hr. Immunoprecipitation combining Western blot analysis showing that a HGD 4 hr treatment could significantly enhanced the tyrosine residue phosphorylation of the Kvl.2. This respon...
|
PDF Full Text Request