Immunohistochemical Determination of Calcium—Calmodulin Binding Predicts Neuronal Damage after Global Ischemia

Abstract

Summ ary: Since ionic Ca 2 + binds with intracellular cal modulin (CaM) before activating proteases, kinases, and phospholipases, demonstration of persistent Ca 2 + -CaM binding in neurons destined to show ischemic cellular in jury would support the concept that elevated intracellular Ca2+ plays a causative role in ischemic neuronal damage. In order to characterize Ca 2 + -CaM binding, we used a sheep anti-CaM antibody (CaM-Ab) which recognizes CaM that is not bound to Ca 2 + or brain target proteins. Therefore, immunohistochemical staining of brain sec tions by labeled CaM-Ab represented only unbound CaM. Six normal rats were compared to 15 animals ren dered 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 second ary antibody was added to stain the CaM-Abo Staining in the endal limb of dentate, dorsal CAl, lateral CA3, and parietal cortex was graded on a 4-point scale. All normal This study describes intraneuronal calcium calmodulin binding in a commonly used in vivo model of global cerebral ischemia. The project was undertaken for two reasons. It is now commonly accepted that calcium entry into neurons and re lease of stored intracellular calcium are pivotal events leading to irreversible cellular damage dur ing the reperfusion phase following an ischemic in sult. In the last several years many laboratories have demonstrated increased calcium in brain and in neurons after cerebral ischemia using a variety of

Protective Effect of DY-9760e, a Calmodulin Antagonist, against Neuronal Cell Death

“…Indeed, it has been previously reported that a persistent increase in the concentration of Ca 2ϩ -bound calmodulin, which is an active form of calmodulin, is associated with ischemic neuronal damage. 13) Furthermore, calmodulin antagonists have been shown to protect against hypoxia/hypoglycemia in organic hippocampal cultures. 14) We have developed a competitive calmodulin antagonist, 3-[2-[4-(3-chloro-2-methylphenylmethyl)-1-piperazinyl]-ethyl]-5,6-dimethoxy-1-(4-imidazolylmethyl)-1H-indazole dihydrochloride 3.5 hydrate (DY-9760e), which exerts a cytoprotective action against the cell death induced by a Ca 2ϩ ionophore 15) and ameliorates brain injury that occurs after transient and permanent focal ischemia in rats.…”

DY-9760e, a Calmodulin Antagonist, Reduces Brain Damage after Permanent Focal Cerebral Ischemia in Cats

“…15,17) DY-9760e is also capable of reducing the ischemic infarct volume when given up to 3 h after the onset of permanent MCA occlusion in rats. 20) Given a previous report showing that the Ca 2ϩ -bound calmodulin complex reaches maximal levels 4 h following cerebral ischemia, it is feasible that the therapeutic time window for DY-9760e is 3 h. 12) Taken together, it is plausible that the amelioration of ischemic brain injury with this drug is mediated by a calmodulin antagonistic action in the brain. Further studies are needed to evaluate the molecular mechanism by which this compound exerts neuroprotective functions.…”

“…There is evidence that calmodulin plays a critical role in ischemic brain injury: (1) a persistent increase of Ca 2ϩ -bound calmodulin, which is an active form, after ischemic insults; (2) significant up-regulation of calmodulin gene expression in the CA1 pyramidal cell layer after cerebral ischemia; and (3) protection by calmodulin antagonists against hypoxic/hypoglycemia in organotypic hippocampal cultures. 12,14,44) Furthermore, there are reports that cells expressing PEP-19, a brain-specific negative regulator of calmodulin, or a mutant calmodulin with Ca 2ϩ -binding defects are resistant to apoptotic stimuli. 45,46) DY-9760e rescues cultured neurons from a wide variety of cell-toxic stimuli that increase intracellular Ca 2ϩ , including excitotoxicity, voltage-gated channel opening, Ca 2ϩ ionophore, and inhibition of endoplasmic reticulum Ca 2ϩ -ATPase.…”

“…In fact, it has been reported that Ca 2ϩ -bound calmodulin, which is an active form, is increased following ischemia and regional changes correlate with regions of ischemic neuronal damage. 12) Furthermore, calmodulin antagonists protect cultured neurons from cell death induced by glutamate, an excitatory amino acid, and protect hippocampal CA1 neurons against hypoxia/hypoglycemia in organotypic cultures. 13,14) DY-9760e (3-[2-[4-(3-chloro-2-methylphenyl)-1-piperazinyl]ethyl]-5,6-dimethoxy-1-(4-imidazolylmethyl)-1H-indazole dihydrochloride 3.5 hydrate) is a calmodulin antagonist that is 2 to 70 times more potent than W-7, a well-known calmodulin antagonist, and can protect neurons against neuronal cell death elicited by Ca 2ϩ overload.…”

Neuronal Protection and Preservation of Calcium/Calmodulin-Dependent Protein Kinase II and Protein Kinase C Activity by Dextrorphan Treatment in Global Ischemia

“…Immunocytochemical staining studies were performed exactly as described previously (Picone et al, 1989).…”