Immunohistochemical determination of calcium-calmodulin binding predicts neuronal damage after global ischemia.
Since ionic Ca2+ binds with intracellular calmodulin (CaM) before activating proteases, kinases, and phospholipases, demonstration of persistent Ca2+-CaM binding in neurons destined to show ischemic cellular injury would support the concept that elevated intracellular Ca2+ plays a causative role in ischemic neuronal damage. In order to characterize Ca2+-CaM binding, we used a sheep anti-CaM antibody (CaM-Ab) which recognizes CaM that is not bound to Ca2+ or brain target proteins. Therefore, immunohistochemical staining of brain sections by labeled CaM-Ab represented only unbound CaM. Six normal rats were compared to 15 animals rendered ischemic for 30 min by a modification of the four-vessel occlusion model. Animals were killed immediately after ischemia, and after 2 and 24 h of reperfusion. Brain sections through hippocampus were incubated in CaM-Ab, and a diaminobenzadiene labeled anti-sheep secondary antibody was added to stain the CaM-Ab. Staining in the endal limb of dentate, dorsal CA1, lateral CA3, and parietal cortex was graded on a 4-point scale. All normal animals had grade 4 staining indicating the presence of unbound CaM in all four brain regions. Ischemic animals demonstrated reduced (grade 0 to 2) staining in the CA1 and CA3 regions immediately and 2 and 24 h after ischemia (p less than 0.01 for both regions at all three time intervals) indicating persistent binding of CaM with Ca2+ and target proteins in these regions. Staining decreased in dentate and cortex up to 2 h after ischemia (p = 0.02 for both regions) but returned toward normal by 24 h.(ABSTRACT TRUNCATED AT 250 WORDS)
Picone
C M
CM
Department of Neurology, University of Texas Medical School, Houston 77030.
Grotta
J C
JC
Earls
R
R
Strong
R
R
Dedman
J
J
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002147:Calmodulin / Q000302:isolation & purification / Q000378:metabolism*; D004533:Egtazic Acid / Q000494:pharmacology; D002546:Ischemic Attack, Transient / Q000378:metabolism* / Q000473:pathology; D008297:Male; D008970:Molecular Weight; D009474:Neurons / Q000378:metabolism* / Q000473:pathology; D011485:Protein Binding; D051381:Rats; D011919:Rats, Inbred Strains; D012016:Reference Values
D002147:Calmodulin; D004533:Egtazic Acid; D002118:Calcium