The role of calcium in apoptosis induced by 7β-hydroxycholesterol and cholesterol-5β,6β-epoxide

Oxysterols, such as 7β-hydroxycholesterol (7β-OH) and cholesterol-5β,6β-epoxide (β-epoxide), may have a central role in promoting atherogenesis. This is thought to be predominantly due to their ability to induce apoptosis in cells of the vascular wall and in monocytes/macrophages. Although there has been extensive research regarding the mechanisms through which oxysterols induce apoptosis, much remains to be clarified. Given that experimental evidence has long associated alterations of calcium (Ca²⁺) homeostasis to apoptotic cell death, the aim of the present study was to determine the influence of intracellular Ca²⁺ changes on apoptosis induced by 7β-OH and β-epoxide. Ca²⁺ responses in differentiated U937 cells were assessed by epifluorescence video microscopy, using the ratiometric dye fura-2. Over 15-min exposure of differentiated U937 cells to 30 μM of 7β-OH induced a slow but significant rise in fura-2 ratio. The Ca²⁺ channel blocker nifedipine and the chelating agent EGTA blocked the increase in cytoplasmic Ca ²⁺. Moreover, dihydropyridine (DHP) binding sites identified with BODIPY-FLX-DHP were blocked following pretreatment with nifedipine, indicating that the influx of Ca²⁺ occurred through L-type channels. However, following long-term incubation with 7β-OH, elevated levels of cytoplasmic Ca²⁺ were not maintained and nifedipine did not provide protection against apoptotic cell death. Our results indicate that the increase in Ca ²⁺ may be an initial trigger of 7β-OH-induced apoptosis, but following chronic exposure to the oxysterol, the influence of Ca²⁺ on apoptotic cell death appears to be less significant. In contrast, Ca ²⁺ did not appear to be involved in β-epoxide-induced apoptosis.