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

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.