Hydrogen Peroxide Inhibits Whole-Cell I Ca,L but Increases on-Cell I Ca,L and Ebselen, an Organoselenium Compound, Suppresses I Ca,L in Arterial Smooth Muscle Cells

To investigate the association between store-operated Ca2+ entry (SOCE) and reactive oxygen species (ROS) during hypoxia, this study determined the changes of transient receptor potential canonical 1 (TRPC1) and Orai1, two candidate proteins for store-operated Ca2+ (SOC) channels and their gate regulator, stromal interaction molecule 1 (STIM1), in a hypoxic environment and their relationship with ROS in pulmonary arterial smooth muscle cells (PASMCs). Exposure to hypoxia caused a transient Ca2+ spike and subsequent Ca2+ plateau of SOCE to be intensified in PASMCs when TRPC1, STIM1, and Orai1 were upregulated. SOCE in cells transfected with specific short hairpin RNA (shRNA) constructs was almost completely eliminated by the knockdown of TRPC1, STIM1, or Orai1 alone and was no longer affected by hypoxia exposure. Hypoxia-induced SOCE enhancement was further strengthened by PEG-SOD but was attenuated by PEG-catalase, with correlated changes to intracellular hydrogen peroxide (H2O2) levels and protein levels of TRPC1, STIM1, and Orai1. Exogenous H2O2 could mimic alterations of the interactions of STIM1 with TRPC1 and Orai1 in hypoxic cells. These findings suggest that TRPC1, STIM1, and Orai1 are essential for the initiation of SOCE in PASMCs. Hypoxia-induced ROS promoted the expression and interaction of the SOC channel molecules and their gate regulator via their converted product, H2O2.

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Potassium currents in cultured human pulmonary arterial smooth muscle cells

Journal of Applied Physiology ◽

10.1152/jappl.1996.80.4.1187 ◽

1996 ◽

Vol 80 (4) ◽

pp. 1187-1196 ◽

Cited By ~ 31

Author(s):

W. Peng ◽

S. V. Karwande ◽

J. R. Hoidal ◽

I. S. Farrukh

Keyword(s):

Smooth Muscle ◽

Smooth Muscle Cells ◽

Single Channel ◽

Muscle Cells ◽

Arterial Smooth Muscle ◽

Whole Cell ◽

Low Concentrations ◽

Pulmonary Arterial ◽

Arterial Smooth Muscle Cells ◽

Pulmonary Arterial Smooth Muscle

In this study, using whole cell and single-channel configurations of the patch-clamp technique, we characterized K+ currents (IK) in cultured human pulmonary arterial smooth muscle cells. The net whole cell outward membrane current (IKo) was activated at potentials positive to -60 mV. One component of IKo, IK(dr), was inhibited by 4-aminopyridine (4-AP) and high concentrations of tetraethylammonium (TEA) but was Ca2+ and charybdotoxin (CTX) insensitive. The other component of IKo, IK(Ca), was voltage and Ca2+ dependent and was inhibited by CTX and low concentrations of TEA. Activation of IKo in single-channel recordings was voltage dependent and demonstrated a high-conductance channel (245 +/- 2 pS) that was Ca2+ and CTX sensitive [IK(Ca)] and a low-conductance channel (109 +/- 2 pS) that was inhibited by 4-AP [IK(dr)] but was insensitive to low concentrations of TEA or to an increase in intracellular [Ca2+]. In isolated pulmonary arterial rings, TEA and 4-AP caused an additive increase in arterial tension. To our knowledge these data provide the first characterization of the IK in human pulmonary arterial smooth muscle cells and indicate that IK(Ca) and IK(dr) play an important role in maintaining pulmonary vascular tone. The data confirm previous observations in pulmonary smooth muscle cells of animal models.

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

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00323.2006 ◽

2007 ◽

Vol 292 (6) ◽

pp. L1598-L1608 ◽

Cited By ~ 58

Author(s):

Mo-Jun Lin ◽

Xiao-Ru Yang ◽

Yuan-Ning Cao ◽

James S. K. Sham

Keyword(s):

Hydrogen Peroxide ◽

Smooth Muscle ◽

Smooth Muscle Cells ◽

Muscle Cells ◽

Channel Blockers ◽

Arterial Smooth Muscle ◽

Intracellular Ca ◽

Pulmonary Arterial ◽

Arterial Smooth Muscle Cells ◽

Pulmonary Arterial Smooth Muscle

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 H2O2 elicited 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. H2O2 did not alter the rate of Mn2+ quenching of fura 2, suggesting store- and receptor-operated Ca2+ channels were not involved. By contrast, H2O2 elicited a sustained inward current carried by Na+ at −70 mV, and the current was inhibited by Ni2+. These results suggest that H2O2 mobilizes 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.

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