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

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

Affiliations
  • 1Department of Physiology and Biophysics, Seoul National University College of Medicine, Seoul 110-799, Korea. insuk@plaza.snu.ac.kr

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 (20microM), 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 (30microM) or an ER Ca2+ pump inhibitor, thapsigargin (1microM), 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 (10microM) 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.

Keyword

Oxidant; DTBNP; DTT; Bovine aortic endothelial cells; Endoplasmic reticulum; Mitochondria

MeSH Terms

Adenosine Triphosphate
Carbonyl Cyanide m-Chlorophenyl Hydrazone
Egtazic Acid
Endoplasmic Reticulum
Endothelial Cells*
Fura-2
Mitochondria
Thapsigargin
Adenosine Triphosphate
Carbonyl Cyanide m-Chlorophenyl Hydrazone
Egtazic Acid
Fura-2
Thapsigargin
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