Abstract 5514: Laminar Shear Stress Upregulates Endothelial KCa2.3 and KCa3.1 Calcium-activated Potassium Channels via Activating Calmodulin-dependent Protein Kinase Kinase and AMP-activated Protein Kinase
Aim Endothelial cell (EC) KCa2.3 and KCa3.1, calcium-activated potassium channels, play a crucial role in NO- and EDHF-mediated regulation of vascular tone by controlling membrane potential and intracellular calcium levels. Since laminar shear stress (LS) is a major determinant of EC function in a calcium-dependent manner, we hypothesized that LS upregulates KCa2.3 and KCa3.1 via activating calcium/calmodulin (CaM) kinase cascade.
Methods Human coronary artery ECs were exposed to static, LS (15 dyn/cm2) or oscillatory shear stress (OS; +/−5 dyn/cm2 × 1Hz) condition for 24 hours in the absence or presence of STO-609 (CaM-dependent kinase kinase (CaMKK) inhibitor, 10μg/ml), Compound C (AMP-activated protein kinase (AMPK) inhibitor, 10μM), KN-62 (calcium/CaM-dependent kinase (CaMK) inhibitor, 10μM), KG-501 (cAMP response element binding protein (CREB) inhibitor, 25μM), or tumor necrosis factor-α (TNF-α; 1 or 10 ng/ml). The mRNA expression and CREB phosphorylation were determined by real-time PCR or Western blotting.
Results LS markedly increased KCa2.3 and KCa3.1 expressions (3.3±0.4 and 6.6±1.8 fold increases vs. static, n=4 –10, p<0.001), whereas OS reduced KCa2.3 and unchanged KCa3.1 expressions (0.4±0.04 and 1.1±0.5 fold, n=3, p=ns). STO-609 significantly inhibited LS-induced increase in KCa2.3 and KCa3.1 expressions (1.8±0.1 fold and 1.5±0.4 fold) and markedly decreased LS-induced CREB phosphorylation (LS; 2.3 fold and LS+STO-609; 1.5 fold vs. static). KCa2.3 and KCa3.1 expressions were also reduced by Compound C (2.2±1.6 and 2.0±1.0 fold, respectively), but not by KN-62 (5.9±1.1 and 6.5±1.2 fold, respectively). KG-501 suppressed LS-induced increase in both channels (KCa2.3; 1.5±0.7 and KCa3.1; 1.9±0.7 fold). TNF-α completely inhibited both channel expressions in a dose-dependent manner (1 and 10 ng/ml; KCa2.3; 0.6±0.01 and 0.1±0.1 fold, and KCa3.1; 0.7±0.1 and 0.3±0.03 fold).
Conclusions LS upregulates KCa2.3 and KCa3.1 expressions via CREB phosphorylation through activation of CaMKK and AMPK, a downstream kinase of CaMKK, whereas TNF-α suppresses both channel expressions. Thus KCa2.3 and KCa3.1 may play an important role in endothelial adaptation to hemodynamic changes and in the maintenance of EC function.