The Effects Of Serum Components On Astrocytic Glutamate Transport

Cerebral hemorrhage is a common and frequently-occurring disease, associated with high morbidity and mortality that severely affects human health. Hematoma and injuries to the surrounding cells are the most important pathological manifestation of cerebral hemorrhage. In addition, epilepsy is often induced by cerebral hemorrhage.In the brain, astrocytes outnumber neurons by at least 5 folds. Astrocytes play important roles in the development and maintenance of normal brain function, particularly in maintaining glutamate homeostasis in the brain that is key to glutamatergic neurotransmission. Compromised astrocytic glutamate transport has been linked to epilepsy and excitotoxic neuronal injuries. In this study, we focus on the influence of cerebral hemorrhage on astrocytic glutamate transport.Purposes Existing studies on cerebral hemorrhage primarily addressed its impact on neuronal activities, with much less attentions paid to astrocytes. Intrigued by the high occurrence rate of epilepsy accompanying cerebral hemorrhage, either in acute phase or weeks after the hemorrhage.Because astrocytes play pivotal roles in glutamate homeostasis in the brain and compromise of astrocyte glutamate uptake often lead to epilepsy and neuronal injury. This study explored the impact of serum components on astrocyte glutamate transport, inspired by some initial observation showing that bovine serum albumin can significantly elevate the equilibrium glutamate concentrations in astrocyte cultures.Methods Cell culture technique, high performance liquid chromatography(HPLC),cellular component fractionation, western blot and immunofluorescence microscopy were used to determine glutamate concentration and expression of glutamate transporters. BSA obtained from different vendors and serums from several species,including human, were tested and compared for their effects on astrocyte glutamate transport.Results BSA(from Fisher) dose-dependently increased the equilibrium [Glu]o in astrocyte cultures,reaching plateau effects at concentrations above 1%. Blocking glutamate transport with TBOA revealed that BSA modulation of equilibrium [Glu]o is primarily due to increase of glutamate release instead of reduction of glutamate uptake.Cellular component fractionation and western blot showed increased plasma membrane expression of cystine-glutamate exchanger(x CT) in astrocytes treated with BSA.Further studies comparing BSA from different vendors indicated that the observed effects on astrocytic equilibrium [Glu]o is unlikely due to BSA per se, but other blood components isolated with the BSA during commercial BSA preparation. Then we focused our studies on the whole serum, which is more relevant to cerebral hemorrhage than impure BSA. As 10% supplement to DMEM culture medium, adult bovine serum,new born calf serum, adult goal serum, adult guinea pig serum and adult human serum all led to elevated equilibrium [Glu]o several folds higher than those in serum free DMEM or DMEM supplemented by 10% fetal bovine serum. These results indicate that serum components can have profound impacts on setting the equilibrium [Glu]o,cerebral hemorrhage can thus chronically elevate equilibrium [Glu]o in the brain to pathologically levels that induce or facilitate epilepsy.Conclusions Astrocyte glutamate uptake and glutamate release, either from astrocytes,or from other sources reach equilibrium when the rate of glutamate uptake equals to glutamate release. Cystine-glutamate exchangers-mediated glutamate release from astrocytes contribute significantly to the setting of equilibrium [Glu]o. Serum components-induced elevation of astrocyte equilibrium [Glu]o provides a mechanistic explanation for epilepsy and neuronal damage frequently associated with cerebral hemorrhage.