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

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 μM–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.