Lysophosphatidylcholine Modulation of Aortic Endothelial Cell Function and the Role of Calcium-Activated Potassium Channels

Caroline Heard, Ian Fearon, Alison Gurney. University of Manchester, Manchester, United Kingdom.

Oxidised low density lipoproteins (oxLDLs) accumulate in atherosclerotic lesions & trigger endothelial dysfunction, supporting the progression of the pathological state. Lysophosphatidylcholine (LPC) is largely accepted as the major biologically active component of oxLDL. This compound is known to modulate the activity of a number of signalling proteins, including Ca2+-activated potassium channels (KCa)1. The aim of this study was to determine LPC activity upon KCa channels expressed within the endothelium & it’s implications for vascular function. A combination of wire myography, electrophysiology & Ca2+ fluorescent imaging were employed to achieve this. P<0.05 was considered significant.

Exposure of phenylephrine-precontracted thoracic aortic rings to LPC (0.5–100μM) induced a dose-dependent relaxation. Control LPC tests (5μM) were compared to those in the presence of various antagonists. The LPC-dilatory effect could be completely abolished with removal of the endothelial layer (control: 21±2%; denuded: -1±1%; n = 4, P<0.05) & was dependent upon influx of Ca2+, as application of La3+ (control: 22±3%; 100μM La3+: 6±4%; n = 4), ruthenium red (control: 30±7%; 50μM RuR: 17±3%; n = 5) & 2-APB (control: 12±1%; 10μM 2-APB: 4±1%; n = 4) attenuated LPC-responses (all P<0.05). The LPC-induced dilation was only attenuated with high concentrations of KCa channel antagonists, TRAM-34 (30μM) & apamin (300nM) (control: 14±2%; KCa blockers: 8±1%; n = 5, P<0.05).

Rat aortic endothelial cells (RAEC) were isolated using the matrigel technique2, & used for patch-clamp & Ca2+-imaging studies. LPC (5μM) caused a hyperpolarisation of RAEC membrane potential (-30±5mV; n = 8, P<0.05) that was sensitive to 500μM TEA+ (-6±3mV; n = 5, P<0.05). Similar responses were induced in HEK293 cells stably transfected with the small KCa (-17±3mV; n = 4, P<0.05) and intermediate KCa channels (-23±4mV; n = 2, not statistically tested). No change in membrane potential was detected in wild-type HEK293 cells treated with LPC (n = 9, P>0.05).

Both wild-type HEK293 cells & RAEC produced robust Ca2+-signalling when treated with LPC (0.5–100μM) as determined by fura-2 AM imaging. In RAEC, 5μM LPC increased the fluorescence ratio (340nm/380nm) from 0.5±0.1 to 1.6±0.3 (n = 6, P<0.05). La3+ (10μM) reduced the LPC-sensitivity as the ratio increased from 0.45±0.01 to only 0.54±0.02 (n = 7, P<0.05). Lowering the [Ca2+] of the perfusing PSS from 1.8mM to 0.5mM also attenuated RAEC LPC-sensitivity, as the fura-2 AM ratio changed from 0.45±0.01 to just 0.57±0.03 (n = 5, P<0.05). Markedly similar responses were detected in wild-type HEK293 cells.

Together these data imply that KCa channel activity is not necessary for the LPC-induced responses and that KCa activation is only secondary to Ca2+ influx. LPC does not directly activate KCa channels.

1. Wolfram Kuhlman et al (2004) J. Mol. Cell. Cardiol. 36: 675-82. 2. Suh et al. (1999) Pflugers. Arch. 438: 612-620.