Suppressive effect of a chloride bicarbonate exchanger inhibitor on staurosporine-induced apoptosis of endothelial cells.

We examined staurosporine-induced apoptosis of vascular endothelial cells with respect to ion movement. Cultured vascular endothelial cells from bovine carotid arteries were used. Apoptosis was determined by propidium iodide assay. Treatment of the endothelial cells (EC) with staurosporine (1 mumol/l) for 6 h induced nuclear fragmentation in 70.5 +/- 1.5% of cells. Concomitant treatment of ECs with 1 mmol/l of 4,4-diisothiocyanatostilbene-2,2-disulfonic acid (DIDS), a chloride bicarbonate exchange blocker, completely inhibited staurosporine-induced apoptosis. Next, we determined whether chloride ion or bicarbonate is involved in apoptosis. Incubation with a chloride ion removal buffer did not inhibit staurosporine-induced apoptosis of ECs; in fact, the buffer itself induced apoptosis in 30.6 +/- 3.3% of cells. Apoptosis due to chloride ion removal was completely inhibited by DIDS (1 mmol/l). Moreover, EC apoptosis due to staurosporine and due to chloride ion (Cl-) removal was completely suppressed by an inhibitor of Ced-3/interleukin 1 beta-converting enzyme (ICE) proteases, benzyloxycarbonyl -Asp- CH2- O(C)O-dichlorobenzene (zD-dcb; 50 mumol/l). Staurosporine (1 mumol/l) increased the intracellular pH of ECs, and DIDS (1 mmol/l) inhibited the increase in pH caused by staurosporine. Our findings suggest that EC apoptosis induced by staurosporine is associated with Cl- and bicarbonate ion (HCO3-). Thus, Cl- efflux from cells or HCO3- influx to cells (which increases pH) may play an important role in signal transduction, such as activation of Ced-3/ICE proteases in staurosporine-induced apoptosis.