Object: Oxyhemoglobin (OxyHb) released from hemolysed erythrocytes has been considered to be responsible for cerebral vasospasm after subarachnoid hemorrhage. The authors previously reported that OxyHb produced apoptosis in cultured vascular endothelial cells. The change in intracellular Ca++ homeostasis was expected to be one of the possible mechanisms of the cytotoxic effects of OxyHb. This study was undertaken to investigate the protective effects of Ca++ channel blockers on OxyHb-induced apoptosis.
Methods: Cultured bovine coronary artery and brain microvascular endothelial cells (passages 5-9) were used. A cell density study, immunohistochemical staining, and DNA fragmentation analysis were performed to confirm apoptosis. Various concentrations (1-50 microM) of OxyHb were used for 24- to 72-hour incubations with and without Ca++-channel blockers. Oxyhemoglobin produced cytotoxicity leading to cell detachment from the culture dish in time- and concentration-dependent manners. The highest dose (50 microM) of OxyHb produced cell detachment after a 24-hour incubation, and the lower doses (1-10 microM) produced cell detachment after 48 to 72 hours. Immunohistochemical analysis showed that apoptosis occurred in cells that were still attached to the side of the culture dish after 48 to 72 hours of OxyHb treatment (5 microM). The OxyHb (10 microM) produced DNA ladders at 48 to 72 hours. Three Ca++-channel blockers were used to prevent the toxic effect of OxyHb. The voltage-dependent Ca++-channel blocker nicardipine (1 microM), the voltage-independent Ca++-channel blocker econazole (10 microM), and the inorganic Ca++-channel blocker lanthanum (100 microM) all failed to prevent cell detachment or DNA ladders produced by OxyHb. These results were similar in both cell lines.
Conclusions: Oxyhemoglobin produced apoptotic changes in cultured vascular endothelial cells, and Ca++-channel blockers did not prevent OxyHb-induced apoptosis.