Traumatic injury of cultured astrocytes alters inositol (1,4,5) trisphosphate-mediated signaling

Our previous studies using an in vitro model of traumatic injury have shown that stretch injury of astrocytes causes a rapid elevation in intracellula r free calcium ([Ca2+](i)), which returns to near normal by 15 min postinju ry. We have also shown that after injury astrocyte intracellular calcium st ores are no longer able to release Ca2+ in response to signal transduction events mediated by the second messenger inositol (1,4,5)-trisphosphate (IP3 , Rzigalinski et al., 1998). Therefore, we tested the hypothesis that in vi tro injury perturbs astrocyte IP3 levels. Astrocytes grown on Silastic memb ranes were labeled with [H-3]-myo-inositol and stretch-injured. Cells and m edia were acid-extracted and inositol phosphates isolated using anion-excha nge columns. Alter injury, inositol polyphosphate (IP3) levels increased up to 10-fold over uninjured controls. Significant injury-induced increases w ere seen at 5, 15, and 30 min and at 24 and 48 h postinjury. Injury-induced increases in IPx were equivalent to the maximal glutamate and trans-(1S,3R )-1-amino-1,3-cyclopentanedicarboxylic acid-stimulated IPx production, howe ver injury-induced increases in IPx were sustained through 24 and 48 h post injury. Injury-induced increases in IPx were attenuated by pretreatment wit h the phospholipase C inhibitors neomycin (100 muM) or U73122 (1.0 muM). Si nce we have previously shown that astrocyte [Ca2+](i) returns to near basal levels by 15 min postinjury, the current results suggest that IP3-mediated signaling is uncoupled from its target, the intracellular Ca2+ store. Unco upling of IP3-mediated signaling may contribute to the pathological alterat ions seen after traumatic brain injury. GLIA 33:12-23, 2001. (C) 2001 Wiley -Liss, Inc.