Acidic pre-conditioning suppresses apoptosis and increases expression of Bcl-xL in coronary endothelial cells under simulated ischaemia.
Ischaemic pre-conditioning has a powerful protective potential against ischaemia-induced cell death, and acidosis is an important feature of ischaemia and can lead to apoptosis. Here we tested whether pre-conditioning with acidosis, that is, acidic pre-conditioning (APC), may protect coronary endothelial cells (EC) against apoptosis induced by simulated ischaemia. For pre-conditioning, EC were exposed fo 40 min. to acidosis (pH 6.4) followed by a 14-hrs recovery period (pH 7.4) and finally treated for 2 hrs with simulated ischaemia (glucose-free anoxia at pH 6.4). Cells undergoing apoptosis were visualized by chromatin staining or by determination of caspase-3 activity Simulated ischaemia in untreated EC increased caspase-3 activity and the number of apoptotic cell (31.3 +/- 1.3%versus 3.9 +/- 0.6% in control). APC significantly reduced the rate of apoptosis (14.2 +/- 1.3%) and caspase-3 activity. Western blot analysis exploring the under lying mechanism leading to this protection revealed suppression of the endoplasmic reticulum- (reduced cleavage of caspase-12) and mitochondria-mediated (reduced cytochrome C release) pathways of apoptosis. These effects were associated with an over-expression of the anti-apoptotic protein Bcl-xL 14 hrs after APC, whereas no effect on the expression of Bcl-2, Bax, Bak, procaspase-12, reticulum-localized chaperones (GRP78, calreticulin), HSP70, HSP32 and HSP27 could be detected. Knock-down of Bcl-xL by siRNA-treatment prevented the protective effect of APC. In conclusion, short acidic pre-treatment can protect EC against ischaemic apoptosis. The mechanism of this protection consists of suppression of the endoplasmic reticulum- and mitochondria-mediated pathways. Over-expression of the anti apoptotic protein Bcl-xL is responsible for the increased resistance to apoptosis during ischaemic insult.
Kumar
S
S
Abteilung für Klinische Pharmakologie, Ruhr-Universität Bochum, Germany.
Reusch
H P
HP
Ladilov
Y
Y
D000143:Acids / Q000494:pharmacology*; D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D053454:Caspase 12 / Q000378:metabolism; D002478:Cells, Cultured; D003327:Coronary Disease / Q000378:metabolism* / Q000473:pathology*; D045304:Cytochromes c / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000187:drug effects / Q000378:metabolism; D042783:Endothelial Cells / Q000187:drug effects* / Q000378:metabolism*; D005786:Gene Expression Regulation; D008297:Male; D018832:Molecular Chaperones / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics; D051381:Rats; D017208:Rats, Wistar; D051020:bcl-X Protein / Q000235:genetics / Q000378:metabolism*
D000143:Acids; D018832:Molecular Chaperones; D034741:RNA, Small Interfering; D051020:bcl-X Protein; D045304:Cytochromes c; D053454:Caspase 12