Repetition of Apoptosis Induced by Amiloride Derivatives in Human Umbilical Vein Endothelial Cells

ABSTRACT

PURPOSE: Human umbilical vein endothelial cells(HUVECs) play an important role in regulating blood flow by releasing vasoactive substances. It has been reported that endothelial impairment and dysfunction might be a primary cause of placental vascular disease, which is manifested clinically as preeclampsia in mother and intrauterine growth restriction in fetus. Furthermore, the frequency of apoptotic changes is increased in umbilical and placental tissues from growth-restricted pregnancies. However, the various mechanisms of umbilical endothelial cell apoptosis have not been broadly proposed. We investigate the effects of amiloride derivatives on apoptotic death of HUVECs and identify their ionic mechanism. METHODS: HUVECs were purchased from Clonetics, and cultured on endothelial cell growth medium. MTT assay and flow cytometry were used for assessing cytotoxic effect and confirming the apoptosis. Changes in intracellular ion concentrations were measured with specific fluorescent dyes and fluorescence imaging analysis system. RESULTS: Amiloride derivatives elicited cytotoxic effects on HUVECs with dose-dependent manners and the rank order of potency is HMA(IC50 11.2 micrometer), MIA>EIPA>>amiloride. HMA-induced cytotoxicity is dependent on extra- and intracellular pH, that is, increase extra- and intracellular pH augmented the cytotoxic effects of HMA. HMA dose-dependently reduced intracellular major ions, such as K+ and Cl-. Interestingly, the depletion of intracellular ions induced by HMA was also significantly enhanced at alkaline extracellular pH. CONCLUSION: Amiloride derivatives induce apoptosis of HUVECs with dose and pH dependent manners. They reduce intracellular K+ and Cl- concentration, which is also extracellular pH dependent.