Korean J Physiol Pharmacol.  2013 Jun;17(3):181-187. 10.4196/kjpp.2013.17.3.181.

Contradictory Effects of Superoxide and Hydrogen Peroxide on KCa3.1 in Human Endothelial Cells

• Choi S
• Na HY
• Kim JA
• Cho SE
• Suh SH

• KMID: 1429296
• DOI: http://doi.org/10.4196/kjpp.2013.17.3.181

Abstract

Reactive oxygen species (ROS) are generated in various cells, including vascular smooth muscle and endothelial cells, and regulate ion channel functions. KCa3.1 plays an important role in endothelial functions. However, the effects of superoxide and hydrogen peroxide radicals on the expression of this ion channel in the endothelium remain unclear. In this study, we examined the effects of ROS donors on KCa3.1 expression and the K+ current in primary cultured human umbilical vein endothelial cells (HUVECs). The hydrogen peroxide donor, tert-butyl hydroperoxide (TBHP), upregulated KCa3.1 expression, while the superoxide donors, xanthine/xanthine oxidase mixture (X/XO) and lysopho-sphatidylcholine (LPC), downregulated its expression, in a concentration-dependent manner. These ROS donor effects were prevented by antioxidants or superoxide dismustase. Phosphorylated extracellular signal-regulated kinase (pERK) was upregulated by TBHP and downregulated by X/XO. In addition, repressor element-1-silencing transcription factor (REST) was downregulated by TBHP, and upregulated by X/XO. Furthermore, KCa3.1 current, which was activated by clamping cells with 1 microM Ca2+ and applying the KCa3.1 activator 1-ethyl-2-benzimidazolinone, was further augmented by TBHP, and inhibited by X/XO. These effects were prevented by antioxidants. The results suggest that hydrogen peroxide increases KCa3.1 expression by upregulating pERK and downregulating REST, and augments the K+ current. On the other hand, superoxide reduces KCa3.1 expression by downregulating pERK and upregulating REST, and inhibits the K+ current. ROS thereby play a key role in both physiological and pathological processes in endothelial cells by regulating KCa3.1 and endothelial function.

Keyword

Ca(2+)-activated K+ channel; Endothelial cells; Hydrogen peroxide; Superoxide

MeSH Terms

Antioxidants
Benzimidazoles
Constriction
Endothelial Cells
Endothelium
Hand
Human Umbilical Vein Endothelial Cells
Humans
Hydrogen
Hydrogen Peroxide
Ion Channels
Muscle, Smooth, Vascular
Oxidoreductases
Pathologic Processes
Phosphotransferases
Reactive Oxygen Species
Superoxides
tert-Butylhydroperoxide
Tissue Donors
Transcription Factors
Antioxidants
Benzimidazoles
Hydrogen
Hydrogen Peroxide
Ion Channels
Oxidoreductases
Phosphotransferases
Reactive Oxygen Species
Superoxides
Transcription Factors
tert-Butylhydroperoxide

Figure

• 

  Fig. 1 KCa3.1 expression in ROS donor-treated HUVECs. KCa3.1 expression was measured using immunoblot, and relative protein expression was represented as a ratio of the levels in the vehicle-treated group to that in the test group. HUVECs were treated with TBHP (A), X/XO (B), or LPC (C), for 24 h; n=4~7. *p<0.05, **p<0.01 versus vehicle-treated control.

• 

  Fig. 2 KCa3.1 expression in ROS donor-treated HUVECs. KCa3.1 expressions were measured using immunocytochemistry. HUVECs were treated with 50 µM TBHP (A), or 50 µM/50 mU/ml X/XO (B), for 24 h. Immunocytochemistry images show KCa3.1 staining in green, and nuclear staining with DAPI in blue; combined images are also presented.

• 

  Fig. 3 Effects of antioxidants on ROS donor-induced KCa3.1 regulation. HUVECs were treated with vehicle or 100 µM TBHP (A), 100 µM/100 mU/ml X/XO (B), or 100 µM LPC (C), for 24 h, with or without pre-treatment with antioxidants (5 µM tempol, 50 µM tiron, or 10 mM NAC), or SOD (1,000 U/ml). The results are mean±SEM of three independent experiments. **p<0.01 versus TBHP, X/XO or LPC alone.

• 

  Fig. 4 Effect of ROS donors on pERK expression in HUVECs. pERK expression was measured using immunoblot, and relative protein expression was expressed as a ratio of the levels in the vehicle-treated group to that in the test group. HUVECs were treated with TBHP (A), X/XO (B), or LPC (C), for 24 h; n=4~7. *p<0.05, **p<0.01, ***p<0.005 versus vehicle-treated control.

• 

  Fig. 5 Effect of ROS donors on REST expression in HUVECs. REST expression was measured using real-time PCR and the relative expression of mRNA was expressed as a ratio of the levels in the vehicle-treated group to that in the test group. HUVECs were treated with TBHP (A) or X/XO (B) for 24 h; n=4~7. *p<0.05, **p<0.01 versus control.

• 

  Fig. 6 Effect of ROS donors on KCa3.1 current in HUVECs. KCa3.1 current was activated by loading cells with 1 µM Ca2+ by using a patch pipette, and treating the cells with 1-EBIO (100 µM). The KCa3.1 currents were normalized to cell capacitance, and the TRAM-34-sensitive current was measured as the KCa3.1 current. (A, B, D, F) Current densities are shown at a membrane potential of +50 mV, and marked by open circles, while I/V relationships were obtained at the points marked by closed circles. (C, F) KCa3.1 current densities at +50 mV; n=6~8 (right panel). *p<0.05 versus control.

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