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 intracellular free calcium ([Ca2+]i), which returns to near normal by 15 min postinjury. We have also shown that after injury astrocyte intracellular calcium stores 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 vitro injury perturbs astrocyte IP3 levels. Astrocytes grown on Silastic membranes were labeled with [3H]-myo-inositol and stretch-injured. Cells and media were acid-extracted and inositol phosphates isolated using anion-exchange columns. After injury, inositol polyphosphate (IPx) levels increased up to 10-fold over uninjured controls. Significant injury-induced increases were 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, however injury-induced increases in IPx were sustained through 24 and 48 h postinjury. Injury-induced increases in IPx were attenuated by pretreatment with the phospholipase C inhibitors neomycin (100 microM) or U73122 (1.0 microM). Since 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. Uncoupling of IP3-mediated signaling may contribute to the pathological alterations seen after traumatic brain injury.
Floyd
C L
CL
Department of Psychology, Medical College of Virginia/Virginia Commonwealth University, Richmond, Virginia 23298, USA.
Rzigalinski
B A
BA
Weber
J T
JT
Sitterding
H A
HA
Willoughby
K A
KA
Ellis
E F
EF
D000818:Animals; D000831:Animals, Newborn; D001253:Astrocytes / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D001930:Brain Injuries / Q000378:metabolism* / Q000503:physiopathology*; D002478:Cells, Cultured / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D003515:Cycloleucine / Q000031:analogs & derivatives* / Q000494:pharmacology; D004963:Estrenes / Q000494:pharmacology; D005110:Extracellular Space / Q000378:metabolism; D018698:Glutamic Acid / Q000378:metabolism / Q000494:pharmacology; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism*; D009355:Neomycin / Q000494:pharmacology; D018696:Neuroprotective Agents / Q000494:pharmacology; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D011760:Pyrrolidinones / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D015398:Signal Transduction / Q000502:physiology*; D013314:Stress, Mechanical; D010738:Type C Phospholipases / Q000378:metabolism
D004963:Estrenes; D018696:Neuroprotective Agents; D010726:Phosphodiesterase Inhibitors; D011500:Protein Synthesis Inhibitors; D011760:Pyrrolidinones; D003515:Cycloleucine; C036361:1-amino-1,3-dicarboxycyclopentane; C060229:1-(6-((3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione; D018698:Glutamic Acid; D015544:Inositol 1,4,5-Trisphosphate; D010738:Type C Phospholipases; D009355:Neomycin