P279	The role of small-conductance calcium-activated potassium channels in endothelial functions of vascular endothelial growth factor.
S.Sultan, V.Sofra, A.Stannard, H.Jia, I.Zachary
British Heart Foundation Laboratories, Centre for Cardiovascular Biology and Medicine, Department of Medicine, University College London, London, GB.

Calcium-activated potassium channels (KCa) have been implicated in maintaining the electrochemical gradient for calcium entry. We investigated the possible role of KCa channels (3 types; BKCa, IKCa, SKCa), particularly the small-conductance KCa channels (SKCa) channel, SK2, in VEGF signalling and regulated functions. Affymetrix arrays hybridised with RNA (VEGF-treated and control HUVECs) showed a significant 8-fold increase of VEGF-induced SK2 expression at 6h (P<0.05). Immunoblotting showed that SK2 protein increased after 1h treatment with VEGF and remained above basal levels at 48h. The intracellular mechanisms mediating VEGF-induced SK2 expression were investigated with the use of signalling inhibitors. Results revealed SK2 expression was inhibited by GF109203 (PKC) and SU5614 (KDR). The role of these channels in VEGF-induced migration was studied using specific channel inhibitors: apamin, charybdotoxin and iberiotoxin. Addition of apamin, charybdotoxin or iberiotoxin inhibited migration of HUVECs induced by VEGF. Inhibitors were evaluated for their effects on an in-vitro assay for endothelial cell-derived tubule development. VEGF induced a 5-fold increase in endothelial cell tubules. Addition of apamin (not charybdotoxin or iberiotoxin) significantly inhibited angiogenesis (P<0.05). In conclusion, we show that VEGF upregulates SK2 expression in HUVECs and identify a novel role for SKCa channels in VEGF-induced endothelial cell migration and angiogenesis. This work was sponsored by a grant from the British Heart Foundation.

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