title: Amiloride inhibits hydrogen peroxide-induced Ca2+ responses in human CNS pericytes.
abstract: The aims of the present study were to investigate the mechanisms of Ca(2+) signaling caused by hydrogen peroxide in CNS pericytes. In cultured human brain microvascular pericytes, cytosolic Ca(2+) 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 microM-10 mM) increased cytosolic Ca(2+) in human CNS pericytes. Cytosolic Ca(2+) remained high after wash-out of hydrogen peroxide. However, the addition of dithiothreitol rapidly reversed cytosolic Ca(2+) to the resting level. The hydrogen peroxide-induced Ca(2+) increase was not inhibited by nicardipine, Gd(3+), La(3+), or omission of external Ca(2+). Neither thapsigargin nor carbonyl cyanide 4-trifluoromethoxyphenylhydrazone attenuated the hydrogen peroxide-induced Ca(2+) rise. Amiloride and its derivatives, benzamil and hexamethylene amiloride reversed the hydrogen peroxide-induced Ca(2+) increase. Human CNS pericytes expressed acid sensing ion channel (ASIC) 1a, Na(+)/Ca(2+) 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 Ca(2+) increase. Hydrogen peroxide releases Ca(2+) from intracellular Ca(2+) pool via an amiloride-sensitive protein, which is controlled by oxidation of thiol group in human CNS pericytes.
authors.affiliation: Department of Medicine and Clinical Science, Graduate School of Medical Sciences, Kyushu University, Maidashi 3-1-1, Higashi-ku, Fukuoka 812-8582, Japan.
authors.forename: Kuniyuki
authors.initials: K
authors.lastname: Nakamura
authors.forename: Masahiro
authors.initials: M
authors.lastname: Kamouchi
authors.forename: Takanari
authors.initials: T
authors.lastname: Kitazono
authors.forename: Junya
authors.initials: J
authors.lastname: Kuroda
authors.forename: Yuji
authors.initials: Y
authors.lastname: Shono
authors.forename: Noriko
authors.initials: N
authors.lastname: Hagiwara
authors.forename: Tetsuro
authors.initials: T
authors.lastname: Ago
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Ooboshi
authors.forename: Setsuro
authors.initials: S
authors.lastname: Ibayashi
authors.forename: Mitsuo
authors.initials: M
authors.lastname: Iida
mesh_terms: D062565:Acid Sensing Ion Channels; D000584:Amiloride / Q000031:analogs & derivatives / Q000494:pharmacology*; D001921:Brain / Q000098:blood supply; D002118:Calcium / Q000378:metabolism; D020013:Calcium Signaling / Q000187:drug effects*; D027682:Cation Transport Proteins / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D003600:Cytosol / Q000737:chemistry / Q000378:metabolism; D004229:Dithiothreitol / Q000494:pharmacology; D004336:Drug Antagonism; D020868:Gene Silencing; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism; D016877:Oxidants / Q000494:pharmacology*; D020286:Pericytes / Q000187:drug effects* / Q000378:metabolism; D026941:Sodium Channel Blockers / Q000494:pharmacology*; D015222:Sodium Channels / Q000235:genetics / Q000378:metabolism; D019831:Sodium-Calcium Exchanger / Q000235:genetics / Q000378:metabolism; D000074082:Sodium-Hydrogen Exchanger 1; D017923:Sodium-Hydrogen Exchangers / Q000235:genetics / Q000378:metabolism
chemical_list: D062565:Acid Sensing Ion Channels; D027682:Cation Transport Proteins; D009419:Nerve Tissue Proteins; D016877:Oxidants; C490998:SLC9A1 protein, human; C429538:SLC9A7 protein, human; D026941:Sodium Channel Blockers; D015222:Sodium Channels; D019831:Sodium-Calcium Exchanger; D000074082:Sodium-Hydrogen Exchanger 1; D017923:Sodium-Hydrogen Exchangers; C110374:sodium-calcium exchanger 1; D000584:Amiloride; D006861:Hydrogen Peroxide; D002118:Calcium; D004229:Dithiothreitol