Mechanisms of oxidative stress-induced calcium signaling in human CNS pericytes

Masahiro Kamouchi, Takanari Kitazono, Junya Kuroda, Kuniyuki Nakamura, Yuji Shono, Noriko Hagiwara, Hiroaki Ooboshi, Setsuro Ibayashi, Mitsuo Iida

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Background and aims: Central nervous system (CNS) pericytes encircle abluminal surface of endothelial cells in brain microvasculature, such as capillaries, arterioles and venules where smooth muscle cells are sparse or lacking. The pericytes closely communicate with the endothelial cells and play a pivotal role in angiogenesis, vascular stability, vascular permeability and regulation of blood brain barrier. We have reported previously that oxidative stress may cause dysregulation of intracellular Ca2+ in rat CNS pericytes. The aims of the present study were to elucidate the mechanisms of reactive oxygen species (ROS)-induced Ca2+ dysregulation in the pericytes. Methods: Using cultured human brain microvascular pericytes, cytosolic Ca2+ concentration was measured by means of fura-2 fluorescence. Production of ROS was detected by fluorescence of dihydroethidium. Reverse transcription and polymerase chain reaction (RT-PCR) was used to examine expression of mRNA. Knockdown of Na+/H+ exchanger1 (NHE1) was done by transfecting the cells with a specific double-strand siRNA for NHE1. Results: Dihydroethidium fluorescence revealed that human CNS pericytes produced ROS at rest without stimulation. Externally applied hydrogen peroxide dose-dependently (10μM-10mM) increased cytosolic Ca2+ in human CNS pericytes. Cytosolic Ca2+ remained high after wash out of hydrogen peroxide; however, addition of dithiothreitol (5mM) rapidly reversed cytosolic Ca2+ to the resting level, indicating that the Ca2+ increase is related to oxidation of thiol. Hydrogen peroxide-induced Ca2+ increase was not inhibited by nicardipine (1μM), Gd3+ (10μM), La3+ (10μM) or omission of external Ca2+. Neither thapsigargin (1μM), an inhibitor of sarcoplasmic/endoplasmic reticulum Ca2+-ATPase, nor carbonyl cyanide 4-trifluoromethoxyphenylhydrazone (10μM), a mitochondria uncoupler, could attenuate the Ca2+ rise. Amiloride (3-30μM) and its derivatives, benzamil (3-30μM) or hexamethylene amiloride (3-30μM) inhibited hydrogen peroxide-induced Ca2+ increase. Human CNS pericytes expressed Na+/Ca2+ exchanger1 (NCX1), NHE1 and NHE7. However, removal of external Na+ did not affect the Ca2+ increase. Moreover, neither KB-R 7943 (10μM), selective inhibitor of NCX, nor ablation of NHE1 by RNA interference inhibited hydrogen peroxide-induced Ca2+ increase. Conclusions: Oxidative stress releases Ca2+ from intracellular store sites via oxidation of thiol in human CNS pericytes. Amiloride sensitive protein other than NCX or NHE is involved in the hydrogen peroxide-induced Ca2+ increase. Oxidative stress may exert its actions at least partially via release of intracellular Ca2+ in human CNS pericytes.

Original languageEnglish
Pages (from-to)BP39-02M
JournalJournal of Cerebral Blood Flow and Metabolism
Issue numberSUPPL. 1
Publication statusPublished - Nov 13 2007

All Science Journal Classification (ASJC) codes

  • Neurology
  • Clinical Neurology
  • Cardiology and Cardiovascular Medicine


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