A possible involvement of ion transporter in tumor necrosis factor α and cycloheximide-induced apoptosis of endothelial cells

WE examined the tumor necrosis factor alpha (TNF alpha)induced apoptosis of vascular endothelial cells from the standpoint of ion channels. Cultured vascular endothelial cells from bovine carotid artery were used. Apoptosis was determined by a propidium iodide assay. Treatment of the endothelial cells with TNF alpha and cycloheximide for Gh induced nuclear fragmentation in a TNF alpha dose-dependent manner (1-10 ng/ml). Concomitant treatment of endothelial cells with TNF alpha at a dose of 10 ng/ml and cycloheximide at a dose of 10 mu g/ml elicited endothelial cell apoptosis as high as 23.4+/-4.1% at Gh after administration. However, 10 ng/ml TNF alpha alone elicited a little apoptosis at bh after its administration (% apoptosis=4.1+/-0.8%). Cycloheximide (10 mu g/ml) did not induce apoptosis at all, Concomitant treatment of endothelial cells with 1 mmol/l of 4,4-diisothiocyanatostilbene-2,2-disulfonic acid, which is a chloride bicarbonate exchanger blocker, partially inhibited the TNF alpha and cycloheximide-induced endothelial cell apoptosis, On the other hand, endothelial cell apoptosis due to TNF alpha and cycloheximide was completely inhibited by benzyloxycarbonyl-Asp-CH2OC(O)-2,6-dichlorobenzene (50 mu mol/l), an inhibitor of caspase. Moreover, pyrrolidine dithiocarbanate, an inhibitor of nuclear factor kappa B (NF-kappa B), also suppressed endothelial cell apoptosis induced by TNF alpha and cycloheximide completely. These findings suggest that the endothelial cell apoptosis induced by TNF alpha and cycloheximide is closely related to not only chloride ions, but also both NF-kappa B and caspase activation. That is to say, there is a possibility that chloride ions or bicarbonate (pH) may play an important role in signal transduction such as NF-kappa B and caspase activation in the apoptosis induced by TNF alpha and cycloheximide.