Hydrogen peroxide-induced Ca2+mobilization in pulmonary arterial smooth muscle cells

Abstract

Reactive oxygen species (ROS) generated from NADPH oxidases and mitochondria have been implicated as key messengers for pulmonary vasoconstriction and vascular remodeling induced by agonists and hypoxia. Since Ca²⁺mobilization is essential for vasoconstriction and cell proliferation, we sought to characterize the Ca²⁺response and to delineate the Ca²⁺pathways activated by hydrogen peroxide (H₂O₂) in rat intralobar pulmonary arterial smooth muscle cells (PASMCs). Exogenous application of 10 μM to 1 mM H₂O₂elicited concentration-dependent increase in intracellular Ca²⁺concentration in PASMCs, with an initial rise followed by a plateau or slow secondary increase. The initial phase was related to intracellular release. It was attenuated by the inositol trisphosphate (IP₃) receptor antagonist 2-aminoethyl diphenylborate, ryanodine, or thapsigargin, but was unaffected by the removal of Ca²⁺in external solution. The secondary phase was dependent on extracellular Ca²⁺influx. It was unaffected by the voltage-gated Ca²⁺channel blocker nifedipine or the nonselective cation channel blockers SKF-96365 and La³⁺, but inhibited concentration dependently by millimolar Ni²⁺, and potentiated by the Na⁺/Ca²⁺exchange inhibitor KB-R 7943. H₂O₂did not alter the rate of Mn²⁺quenching of fura 2, suggesting store- and receptor-operated Ca²⁺channels were not involved. By contrast, H₂O₂elicited a sustained inward current carried by Na⁺at −70 mV, and the current was inhibited by Ni²⁺. These results suggest that H₂O₂mobilizes intracellular Ca²⁺through multiple pathways, including the IP₃- and ryanodine receptor-gated Ca²⁺stores, and Ni²⁺-sensitive cation channels. Activation of these Ca²⁺pathways may play important roles in ROS signaling in PASMCs.