Responses of astrocyte to simultaneous glutamate and arachidonic acid treatment.

After cellular injury many endogenous toxins are released from injured cells and result in secondary injury. To elucidate mechanisms of such injury many of these toxins have been studied individually. However, the data obtained is only useful for reference and does not accurately represent the multifactorial situation under pathophysiological conditions. Primary astrocytic cultures were treated individually and simultaneously with two well-studied toxins, glutamate (Glu) and arachidonic acid (AA). Both are simultaneously released from neural cells during injury. Measurements of cellular protein content, intracellular water space, lactate dehydrogenase release, and malondialdehyde formation indicated that Glu and AA act through different mechanisms. Glu+AA applied together had a synergistic effect on the levels of Caspase-3 gene expression, and Bcl-2 and Hsp70 protein. Atomic force microscopy observed that Glu caused cell membrane roughness and nuclear swelling, while AA induced pores in the cell membrane and nuclear shrinkage. Glu+AA accelerated nuclear shrinkage and resulted in more serious cell damage. This study not only distinguishes the different responses of astrocytes to Glu and AA, but also provides a new view into the synergistic effect of these biochemicals; highlighting the need to be cautious in applying single factor experimental data to interpret complex physiological and pathological conditions in animals. Two or more factors may act not only on different targets but also on the same target synergistically.

Ischemia activates JNK/c-Jun/AP-1 pathway to up-regulate 14-3-3gamma in astrocyte.

Ischemia occurs in the brain as the result of stroke and other related injuries and few therapies are effective. If more is understood then potential treatments could be investigated. It was previously reported that 14-3-3gamma could be up-regulated by ischemia in astrocyte to protect cells from ischemia-induced apoptosis. In this study, we attempted to uncover the mechanism responsible for this 14-3-3gamma up-regulation in primary culture of astrocytes under ischemic-like conditions. It was found that in vitro ischemia may activate PI3K/Akt and MAPK signaling pathways. Astrocyte cultures were treated with LY294002 (PI3K inhibitor), U0126 (ERK inhibitor), SB203580 (p38 inhibitor) and SP600125 (JNK inhibitor). Only SP600125 could inhibit the ischemia-induced 14-3-3gamma up-regulation in astrocytes. At the same time, we observed an ischemia-induced nuclear translocation of p-c-Jun, a major downstream component of JNK. Inhibition of AP-1 with curcumin also inhibited 14-3-3gamma up-regulation indicating that ischemia-induced up-regulation of 14-3-3gamma in astrocyte involves activation of the JNK/p-c-Jun/AP-1 pathway.