Intracellular Ca2+ signalling concentration increase induced by 3β-16β, 23, 29-tetrahydroxyoleane-12-ene (THO) on rat aorta endothelial cells.

Ca2+ signalling induced by 3β-16β, 23, 29-tetrahydroxyoleane-12-ene (10-4 M) in in situ endothelium of aortic rings was evaluated to investigated the mechanisms underlying intracellular Ca2+ signalling in rat aortic endothelium loaded with the Ca2+-sensitive dye, fura-2/AM. In situ endothelium cells were visualized by an upright epifluorescence Axiolab microscope. Cytoplasm-free calcium concentration [Ca2+] i was estimated by determining the fluorescence ratio of the [Ca2+] i probes, Fura 2/AM. This study showed that THO caused a slow, long lasting increase in the [Ca2+] i of aortic endothelial cells. Such a slow [Ca2+] i increase was very limited in Ca2+-free extracellular medium. THO, administrated in Ca2+ free medium did not increase intracellular influx Ca2+. What this suggests is that THO would act on the calcium channels by stimulating their openings, thus allowing a massive entry of Ca2+ into the cell. The lack of Ca2+ entry was verified by the failure of ATP to produce increase amplitude in the aortic rings in the absence of extracellular Ca2+. The study  showed that the slow increase in [Ca2+]i did not involve the sarcoplasmic reticulum Ca2+-pump, through pre-treatment of our preparations with Cyclopiazonic acid (10 mM) inhibitor of sarcoplasmic reticulum Ca2+-pump. To determine if THO-induced intracellular influx Ca2+ involves the participation of channels calcium, the preparations were pre-treated with Lanthanum III the non-specific calcium channel antagonists and the result showed that lanthanum III (100 µM) completely abolished the higher amplitude due to THO effect. THO-induced intracellular influx Ca2+ involves the participation of channels calcium.