The Effects of DTBNP on Intracellular Ca<SUP>2+</SUP> Signaling in Cultured Bovine Aortic Endothelial Cells

The Effects of DTBNP on Intracellular Ca<SUP>2+</SUP> Signaling in Cultured Bovine Aortic Endothelial Cells

The mechanism underlying oxidant-induced intracellular Ca<SUP>2+</SUP> ([Ca<SUP>2+</SUP>]<SUB>i</SUB>) increase was studied in cultured bovine aortic endothelial cells (BAECs) using fura-2 AM. In the presence of 2 mM extracellular Ca<SUP>2+</SUP>, the application of DTBNP (20μM), a membrane-permeable oxidant, caused an increase in [Ca<SUP>2+</SUP>]<SUB>i</SUB>, and DTT (2 mM) as a reductant completely reversed the effect of DTBNP. The [Ca<SUP>2+</SUP>]<SUB>i </SUB>increase induced by DTBNP was also observed in an extracellular Ca<SUP>2+</SUP>-free/2 mM EGTA solution, indicating the release of Ca<SUP>2+</SUP> from intracellular store(s). After endoplasmic reticulum was depleted by an IP<SUB>3</SUB>-generating agonist, ATP (30μM) or an ER Ca<SUP>2+</SUP> pump inhibitor, thapsigargin (1μM), DTBNP-stressed BAECs showed an increase of [Ca<SUP>2+</SUP>]<SUB>i</SUB> in Ca<SUP>2+</SUP>-free/2 mM EGTA solution. Ratio-differences before and after the application of DTBNP after pretreatment with ATP or thapsigargin were 0.42&#8273;0.15 and 0.49&#8273;0.07, respectively (n=7), which are significantly reduced, compared to the control value of 0.72&#8273;0.07 in a Ca<SUP>2+</SUP>-free/2 mM EGTA solution. After the protonophore CCCP (10μM) challenge to release mitochondrial Ca<SUP>2+</SUP>, the similar result was obtained. Ratio-difference before and after the application of DTBNP after pretreatment with CCCP was 0.46&#8273;0.09 (n=7). Simultaneous application of thapsigargin and CCCP completely abolished the DTBNP-induced [Ca<SUP>2+</SUP>]<SUB>i</SUB> increase. The above results together indicate that the increase of [Ca<SUP>2+</SUP>]<SUB>i</SUB> by DTBNP resulted from the release of Ca<SUP>2+</SUP> from both endoplasmic reticulum and mitochondria.