Importance of bicarbonate transport for ischaemia-induced apoptosis of coronary endothelial cells.
Bicarbonate transport (BT) has been previously shown to participate in apoptosis induced by various stress factors. However, the precise role of BT in ischaemia-induced apoptosis is still unknown. To investigate this subject, rat coronary endothelial cells (EC) were exposed to simulated ischaemia (glucose free anoxia at Ph 6.4) for 2 hrs and cells undergoing apoptosis were visualized by nuclear staining or by determination of cas-pase- 3 activity. To inhibit BT, EC were either treated with the inhibitor of BT 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS, 300 mumol/l) or exposed to ischaemia in bicarbonate free, 4-(2-hydroxyethyl)-I-piperazi-neethanesulphonic acid (HEPES)-buffered medium. Simulated ischaemia in bicarbonate-buffered medium (Bic) increased caspase-3 activity and the number of apoptotic cell (23.7 + 1.4%versus 5.1 + 1.2% in control). Omission of bicarbonate during ischaemia further significantly increased caspase-3 activity and the number of apoptotic cells (36.7 1.7%). Similar proapoptotic effect was produced by DIDS treatment during ischaemia in Bic, whereas DIDS had no effect when applied in bicarbonate-free, HEPES-buffered medium (Hep). Inhibition of BT was without influence on cytosolic acidification during ischaemia and slightly reduced cytosolic Ca(2+) accumulation. Initial characterization of the underlying mechanism leading to apoptosis induced by BT inhibition revealed activation of the mitochondrial pathway of apoptosis, i.e., increase of cytochrome C release, depolarization of mitochondria and translocation of Bax protein to mitochondria. In contrast, no activation of death receptor-dependent pathway (caspase-8 cleavage) and endoplasmic reticulum- dependent pathway (caspase-12 cleavage) was detected. In conclusion, BT plays an important role in ischaemia-induced apoptosis of coronary EC by suppression of mitochondria-dependent apoptotic pathway.
Kumar
Sanjeev
S
Abteilung für Klinische Pharmakologie, Ruhr-Universität Bochum, Universitätsstrasse 150, D-44801 Bochum, Germany.
Kasseckert
Sascha
S
Kostin
Sawa
S
Abdallah
Yaser
Y
Piper
Hans Michael
HM
Steinhoff
Gustav
G
Reusch
H Peter
HP
Ladilov
Yury
Y
D000138:Acidosis; D000818:Animals; D017209:Apoptosis; D001639:Bicarbonates / Q000378:metabolism*; D001692:Biological Transport; D002118:Calcium / Q000378:metabolism; D053454:Caspase 12 / Q000378:metabolism; D053148:Caspase 3 / Q000378:metabolism; D015687:Cell Hypoxia; D045304:Cytochromes c / Q000378:metabolism; D003600:Cytosol / Q000473:pathology; D042783:Endothelial Cells / Q000201:enzymology / Q000378:metabolism* / Q000473:pathology*; D006863:Hydrogen-Ion Concentration; D008297:Male; D053078:Membrane Potential, Mitochondrial; D008928:Mitochondria; D017202:Myocardial Ischemia / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D013997:Time Factors; D051028:bcl-2-Associated X Protein / Q000378:metabolism
D001639:Bicarbonates; D051028:bcl-2-Associated X Protein; D045304:Cytochromes c; D053454:Caspase 12; D053148:Caspase 3; D002118:Calcium