Korean J Physiol Pharmacol.  2009 Feb;13(1):27-32. 10.4196/kjpp.2009.13.1.27.

Oxidized Low-density Lipoprotein- and Lysophosphatidylcholine-induced Ca2+ Mobilization in Human Endothelial Cells

• Kim MY
• Liang GH
• Kim JA
• Choi SS
• Choi S
• Suh SH

• KMID: 2285368
• DOI: http://doi.org/10.4196/kjpp.2009.13.1.27

Abstract

The effects of oxidized low-density lipoprotein (OxLDL) and its major lipid constituent lysophosphatidylcholine (LPC) on Ca2+ entry were investigated in cultured human umbilical endothelial cells (HUVECs) using fura-2 fluorescence and patch-clamp methods. OxLDL or LPC increased intracellular Ca2+ concentration ([Ca2+]i), and the increase of [Ca2+]i by OxLDL or by LPC was inhibited by La3+ or heparin. LPC failed to increase [Ca2+]i in the presence of an antioxidant tempol. In addition, store-operated Ca2+ entry (SOC), which was evoked by intracellular Ca2+ store depletion in Ca2+-free solution using the sarcoplasmic reticulum Ca2+ pump blocker, 2, 5-di-t-butyl-1, 4-benzohydroquinone (BHQ), was further enhanced by OxLDL or by LPC. Increased SOC by OxLDL or by LPC was inhibited by U73122. In voltage-clamped cells, OxLDL or LPC increased [Ca2+]i and simultaneously activated non-selective cation (NSC) currents. LPC-induced NSC currents were inhibited by 2-APB, La3+ or U73122, and NSC currents were not activated by LPC in the presence of tempol. Furthermore, in voltage-clamped HUVECs, OxLDL enhanced SOC and evoked outward currents simultaneously. Clamping intracellular Ca2+ to 1 micrometer activated large-conductance Ca2+-activated K+ (BKCa) current spontaneously, and this activated BKCa current was further enhanced by OxLDL or by LPC. From these results, we concluded that OxLDL or its main component LPC activates Ca2+-permeable Ca2+-activated NSC current and BKCa current simultaneously, thereby increasing SOC.

Keyword

Oxidized LDL; Endothelial cell; Store-operated Ca2+ entry; Large conductance Ca2+-activated K+ channel; Nonselective cation current

MeSH Terms

Constriction
Cyclic N-Oxides
Endothelial Cells
Estrenes
Fluorescence
Fura-2
Heparin
Humans
Lipoproteins
Lipoproteins, LDL
Lysophosphatidylcholines
Pyrrolidinones
Sarcoplasmic Reticulum
Spin Labels
Cyclic N-Oxides
Estrenes
Fura-2
Heparin
Lipoproteins
Lipoproteins, LDL
Lysophosphatidylcholines
Pyrrolidinones
Spin Labels

Figure

• 

  Fig. 1. Effect of OxLDL or LPC on intracellular Ca2+ concentration ([Ca2+]i) in HUVECs. (A, C and D) OxLDL-induced increase of [Ca2+]i in the presence of (A, C) or in the absence of extracellular Ca2+ (D). (B) in the presence of an antioxidant tempol, LPC failed to increase [Ca2+]i. (E and F) The increase of store-operated Ca2+ entry by OxLDL (E) or LPC (F).

• 

  Fig. 2. Effect of OxLDL on store-operated Ca2+ entry in a voltage-clamped cell. Membrane currents and [Ca2+]i were measured simultaneously at a holding potential of 0 mV in nystatin-perforated mode. (A) further activation of store-operated Ca2+ entry (SOC) by OxLDL. SOC was activated by depletion of the intracellular Ca2+ store and re-exposure to Ca2+. (B) time course of membrane currents at +50 and −50 mV activated by a rise in [Ca2+]i (A). Membrane currents were activated by repetitive ramps from −100 to +100 mV. (C) current-voltage relations obtained at points indicated 1~3 in (B).

• 

  Fig. 3. Effect of LPC on nonselective cation (NSC) currents in HUVECs. Membrane potential was held at 0 mV. (A, C and E) time courses of membrane currents obtained at +50 and −50 mV during repetitive ramps from −100 to +100 mV. (B, D and F) current-voltage relations obtained at points indicated 1~9 in (A), (C) and (E), respectively. (G) bar graph showing current density at +50 mV. n=8, ∗p < 0.001.

• 

  Fig. 4. Effect of OxLDL on store-operated Ca2+ entry and membrane currents in a voltage-clamped cell. Membrane currents (B) and [Ca2+]i (A) were measured simultaneously at a holding potential of 0 mV in nystatin-perforated mode.

• 

  Fig. 5. Effect of OxLDL and LPC on BKCa currents. BKCa currents were activated by clamping [Ca2+]i at 1 (A-C) or 0.5 μM (D). Membrane potential was held at 0 mV. (A and D) time course of membrane current change by OxLDL (A) or LPC (D) measured at −50 and +100 mV. (B) current-voltage relationship obtained from the voltage ramps from −100 to +100 mV labeled as a and b in (A). (C) bar graph showing current density at +100 mV. (C) control. OxLDL, maximal current density obtained during OxLDL (20 μg/ml) application; w/o, wash-out. n=17, ∗p <0.001.

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