Proceedings of The Physiological Society
University of Manchester (2010) Proc Physiol Soc 19, C46
Calcium signalling is altered in aortic endothelial cells of Apolipoprotein E knockout mice.
C. Prendergast1, J. Quayle2, T. Burdyga1, S. Wray1
1. Physiology, University of Liverpool, Liverpool, United Kingdom. 2. Human Anatomy and Cell Biology, University of Liverpool, Liverpool, United Kingdom.
Apolipoprotein E knockout (ApoE-/-) mice develop hypercholesterolemia and are a widely used model of atherosclerosis. An endothelial dysfunction, characterised by reduced ACh-mediated relaxation, has been reported in plaque-laden regions of the aorta and even in plaque-free areas the basal availability of NO appears to be compromised before the onset of disease. Yet little is known about the intracellular Ca2+ signalling of these cells and how it is affected by this hypercholesterolemic environment. Therefore, our aim was to examine the Ca2+ responses of wild type (WT) and ApoE-/- mouse aorta endothelial cells using confocal microscopy, allowing the integrity of the endothelium:vascular smooth muscle interface to remain intact. We have examined whether there is a difference in basal Ca2+ levels between these 2 endothelial cell phenotypes and characterised the [Ca2+] response to the agonists carbachol (CCh) and adenosine triphosphate (ATP) in terms of peak response observed and whether a homogenous response was achieved across all cells. In addition, we determined whether further modulation of plasma membrane cholesterol levels, using methylcyclodextrin (MCD), altered the responses. Data are presented as mean±sem and comparisons made using a Student’s t-test. Basal Ca2+ levels were determined to not be significantly different in aortic endothelial cells from WT and ApoE-/- mice (Fluorescence: 384.5±4.7, n=150 v 397.4±4.4, n=234). Endothelial cells did not respond homogeneously to agonist application. We observed that 61±7% of WT cells responded to ATP and 42±6% to CCh. Similarly, 51±5% of ApoE-/- cells responded to ATP and 34±7% to CCh. Therefore, the high cholesterol environment of the ApoE-/- cells does not alter the baseline characteristics of these cells. The Ca2+ response to both CCh and ATP is characterised by a rapid upstroke, followed by a strongly sustained response in both tissue types. The peak response to CCh stimulation was significantly larger in the ApoE-/- cells (F/F0: 0.97±0.02, n=69 versus 1.21±0.08, n=69, P=0.014), whereas the peak response to ATP stimulation was significantly smaller (F/F0: 1.2±0.05, n=83 versus 0.99±0.04, n=96, P=0.002), demonstrating a clear difference in agonist-mediated Ca2+ signalling in the endothelial cells of the knockout mouse. When WT aortic strips were incubated with 2% MCD for 10min to extract cholesterol from the cell membrane and disrupt caveolae, peak responses to ATP and CCh were unaltered. When ApoE-/- aortic strips were incubated with MCD, the peak response to ATP was unaltered, however, the response to CCh was significantly reduced (F/F0: 1.21±0.08, n=69 v 0.78±0.04, n=45, P>0.0001). We have demonstrated that while baseline Ca2+ and cell responsiveness remain the same, Ca2+ signalling processes are significantly altered in the aortic endothelial cells of the ApoE-/- mouse.
Where applicable, experiments conform with Society ethical requirements