Amiloride inhibits hydrogen peroxide-induced Ca2+ responses in human CNS pericytes

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

Research output: Contribution to journalArticle

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Abstract

The aims of the present study were to investigate the mechanisms of Ca2+ signaling caused by hydrogen peroxide in CNS pericytes. In cultured human brain microvascular pericytes, cytosolic Ca2+ concentration was measured by means of fura-2 fluorescence. Reverse transcription and polymerase chain reaction was performed to examine the expression of mRNA. Knockdown of Na+/H+ exchanger (NHE) was done by transfecting the cells with specific double-strand siRNAs for NHE. Externally applied hydrogen peroxide dose-dependently (100 μM-10 mM) increased cytosolic Ca2+ in human CNS pericytes. Cytosolic Ca2+ remained high after wash-out of hydrogen peroxide. However, the addition of dithiothreitol rapidly reversed cytosolic Ca2+ to the resting level. The hydrogen peroxide-induced Ca2+ increase was not inhibited by nicardipine, Gd3+, La3+, or omission of external Ca2+. Neither thapsigargin nor carbonyl cyanide 4-trifluoromethoxyphenylhydrazone attenuated the hydrogen peroxide-induced Ca2+ rise. Amiloride and its derivatives, benzamil and hexamethylene amiloride reversed the hydrogen peroxide-induced Ca2+ increase. Human CNS pericytes expressed acid sensing ion channel (ASIC) 1a, Na+/Ca2+ exchanger (NCX) 1, Na+/H+ exchanger (NHE) 1, and NHE7. However, the removal of external Na+, treatment with KB-R 7943 and mibefradil, or knockdown of NHE1 and NHE7 did not affect the hydrogen peroxide-induced Ca2+ increase. Hydrogen peroxide releases Ca2+ from intracellular Ca2+ pool via an amiloride-sensitive protein, which is controlled by oxidation of thiol group in human CNS pericytes.

Original languageEnglish
Pages (from-to)327-334
Number of pages8
JournalMicrovascular Research
Volume77
Issue number3
DOIs
Publication statusPublished - May 1 2009

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Pericytes
Amiloride
Hydrogen Peroxide
Sodium-Hydrogen Antiporter
Acid Sensing Ion Channels
Mibefradil
Nicardipine
Thapsigargin
Fura-2
Dithiothreitol
Polymerase chain reaction
Transcription
Sulfhydryl Compounds
Reverse Transcription
Brain
Fluorescence
Derivatives
Polymerase Chain Reaction
Oxidation
Messenger RNA

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Cardiology and Cardiovascular Medicine
  • Cell Biology

Cite this

Amiloride inhibits hydrogen peroxide-induced Ca2+ responses in human CNS pericytes. / Nakamura, Kuniyuki; Kamouchi, Masahiro; Kitazono, Takanari; Kuroda, Junya; Shono, Yuji; Hagiwara, Noriko; Ago, Tetsuro; Ooboshi, Hiroaki; Ibayashi, Setsuro; Iida, Mitsuo.

In: Microvascular Research, Vol. 77, No. 3, 01.05.2009, p. 327-334.

Research output: Contribution to journalArticle

Nakamura, Kuniyuki ; Kamouchi, Masahiro ; Kitazono, Takanari ; Kuroda, Junya ; Shono, Yuji ; Hagiwara, Noriko ; Ago, Tetsuro ; Ooboshi, Hiroaki ; Ibayashi, Setsuro ; Iida, Mitsuo. / Amiloride inhibits hydrogen peroxide-induced Ca2+ responses in human CNS pericytes. In: Microvascular Research. 2009 ; Vol. 77, No. 3. pp. 327-334.
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