The role of calcium in apoptosis induced by 7beta-hydroxycholesterol and cholesterol-5beta,6beta-epoxide.
Oxysterols, such as 7beta-hydroxy-cholesterol (7beta-OH) and cholesterol-5beta,6beta-epoxide (beta-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 (Ca(2+)) homeostasis to apoptotic cell death, the aim of the present study was to determine the influence of intracellular Ca(2+) changes on apoptosis induced by 7beta-OH and beta-epoxide. Ca(2+) 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 muM of 7beta-OH induced a slow but significant rise in fura-2 ratio. The Ca(2+) 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 Ca(2+) occurred through L-type channels. However, following long-term incubation with 7beta-OH, elevated levels of cytoplasmic Ca(2+) 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 7beta-OH-induced apoptosis, but following chronic exposure to the oxysterol, the influence of Ca(2+) on apoptotic cell death appears to be less significant. In contrast, Ca(2+) did not appear to be involved in beta-epoxide-induced apoptosis.
Lordan
Sinéad
S
Department of Food and Nutritional Sciences, University College, Cork, Republic of Ireland.
O'Brien
Nora M
NM
Mackrill
John J
JJ
D017209:Apoptosis / Q000187:drug effects*; D001562:Benzimidazoles / Q000378:metabolism; D002118:Calcium / Q000378:metabolism*; D002121:Calcium Channel Blockers / Q000494:pharmacology; D016923:Cell Death / Q000187:drug effects; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000378:metabolism; D002614:Chelating Agents / Q000494:pharmacology; D003593:Cytoplasm / Q000737:chemistry / Q000187:drug effects; D004533:Egtazic Acid / Q000494:pharmacology; D004852:Epoxy Compounds / Q000494:pharmacology*; D005456:Fluorescent Dyes / Q000378:metabolism; D016257:Fura-2 / Q000378:metabolism; D006801:Humans; D006888:Hydroxycholesterols / Q000494:pharmacology*; D007770:L-Lactate Dehydrogenase / Q000032:analysis / Q000378:metabolism; D008856:Microscopy, Fluorescence; D018715:Microscopy, Video; D009543:Nifedipine / Q000494:pharmacology; D013997:Time Factors; D020298:U937 Cells
D001562:Benzimidazoles; D002121:Calcium Channel Blockers; D002614:Chelating Agents; D004852:Epoxy Compounds; D005456:Fluorescent Dyes; D006888:Hydroxycholesterols; D004533:Egtazic Acid; C011724:cholest-5-en-3 beta,7 alpha-diol; D007770:L-Lactate Dehydrogenase; D009543:Nifedipine; C017807:bisbenzimide ethoxide trihydrochloride; D002118:Calcium; D016257:Fura-2