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pISSN 1226-4512 eISSN 2093-3827

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Korean J Physiol Pharmacol 2005; 9(6): 341-346

Published online December 30, 2005

Copyright © Korean J Physiol Pharmacol.

The Effects of DTBNP on Intracellular Ca2+ Signaling in Cultured Bovine Aortic Endothelial Cells

Sung Jin Park, Byung Joo Kim, Mei Hong Zhu, Insuk So, and Ki Whan Kim

Department of Physiology and Biophysics, Seoul National University College of Medicine, Seoul 110⁣799, Korea

Abstract

The mechanism underlying oxidant-induced intracellular Ca2+ ([Ca2+]i) increase was studied in cultured bovine aortic endothelial cells (BAECs) using fura-2 AM. In the presence of 2 mM extracellular Ca2+, the application of DTBNP (20μM), a membrane-permeable oxidant, caused an increase in [Ca2+]i, and DTT (2 mM) as a reductant completely reversed the effect of DTBNP. The [Ca2+]i increase induced by DTBNP was also observed in an extracellular Ca2+-free/2 mM EGTA solution, indicating the release of Ca2+ from intracellular store(s). After endoplasmic reticulum was depleted by an IP3-generating agonist, ATP (30μM) or an ER Ca2+ pump inhibitor, thapsigargin (1μM), DTBNP-stressed BAECs showed an increase of [Ca2+]i in Ca2+-free/2 mM EGTA solution. Ratio-differences before and after the application of DTBNP after pretreatment with ATP or thapsigargin were 0.42⁑0.15 and 0.49⁑0.07, respectively (n=7), which are significantly reduced, compared to the control value of 0.72⁑0.07 in a Ca2+-free/2 mM EGTA solution. After the protonophore CCCP (10μM) challenge to release mitochondrial Ca2+, the similar result was obtained. Ratio-difference before and after the application of DTBNP after pretreatment with CCCP was 0.46⁑0.09 (n=7). Simultaneous application of thapsigargin and CCCP completely abolished the DTBNP-induced [Ca2+]i increase. The above results together indicate that the increase of [Ca2+]i by DTBNP resulted from the release of Ca2+ from both endoplasmic reticulum and mitochondria.

Keywords: Oxidant, DTBNP, DTT, Bovine aortic endothelial cells, Endoplasmic reticulum, Mitochondria