Background-Angioplasty

1 Activation of Ca 2+ -activated K + -channels (K Ca ) has been suggested to play a key role in endothelium-derived hyperpolarizing factor (EDHF)-mediated vasodilation. However, due to the low selectivity of commonly used K Ca -channel blockers it is still elusive which endothelial K Casubtypes mediate hyperpolarization and thus initiate EDHF-mediated vasodilation. 2 Using the non-cytochrome P450 blocking clotrimazole-derivatives, 1-[(2-chlorophenyl) diphenylmethyl]-1H-pyrazole (TRAM-34) and 2-(2-chlorophenyl)-2,2-diphenylacetonitrile (TRAM-39) as highly selective IK1-inhibitors, we investigated the role of the intermediate-conductance K Ca (rIK1) in endothelial hyperpolarization and EDHF-mediated vasodilation. 3 Expression and function of rIK1 and small-conductance K Ca (rSK3) were demonstrated in situ in single endothelial cells of rat carotid arteries (CA). rIK1-currents were blocked by TRAM-34 or TRAM-39, while rSK3 was blocked by apamin. In current-clamp experiments, endothelial hyperpolarization in response to acetylcholine was abolished by the combination of apamin and TRAM-34. 4 In phenylephrine-preconstricted CA, acetylcholine-induced NO and prostacyclin-independent vasodilation was almost completely blocked by ChTX, CLT, TRAM-34, or TRAM-39 in combination with the SK3-blocker apamin. Apamin, TRAM-34, and CLT alone or sulphaphenzole, a blocker of the cytochrome P450 isoform 2C9, were ine ective in blocking the EDHF-response. 5 In experiments without blocking NO and prostacyclin synthesis, the combined blockade of SK3 and IK1 reduced endothelium-dependent vasodilation. 6 In conclusion, the use of selective IK1-inhibitors together with the SK3-blocker apamin revealed that activation of both K Ca , rIK1 and rSK3 is crucial in mediating endothelial hyperpolarization and generation of the EDHF-signal while the cytochrome P450 pathway seems to play a minor or no role in rat CA.

Objective-Ca2ϩ -activated K ϩ (K Ca ) channels have been proposed to promote mitogenesis in several cell types. Here, we tested whether the intermediate-conductance K Ca channel (IKCa1) and the large-conductance K Ca channel (BK Ca ) contribute to endothelial cell (EC) proliferation and angiogenesis. Material and Results-Function and expression of IKCa1 and BK Ca /Slo were investigated by patch-clamp analysis and real-time RT-PCR in human umbilical vein ECs (HUVECs) and in dermal human microvascular ECs 1 (HMEC-1 ngiogenesis is an important process in a variety of physiological and pathophysiological conditions such as embryonic vasculogenesis, wound healing, poststenotic collateral formation, inflammation, and tumor vascularization. 1 Regarding the latter, tumor-derived angiogenic factors such as the vascular endothelial growth factor (VEGF) and the basic fibroblast growth factor (bFGF) induce endothelial cell (EC) sprouting, proliferation, migration, and finally new vessel formation. 1,2 With respect to EC proliferation, several studies have shown that EC proliferation after stimulation with angiogenic factors is initiated by a rise in intracellular calcium mediated through calcium-influx channels. [3][4][5] By keeping the membrane potential negative, chloride and potassium channels provide the driving force for this calcium entry and thus play an important role in regulating cell cycle progression 6 -9 as well as angiogenesis, as shown previously for chloride channels. 6 In particular, the intermediate- Materials and Methods Human ECs and In Vitro Proliferation StudiesHUVECs were isolated as described previously. 23 HUVECs and HMEC-1, a cell line derived from dermal HMECs 24 were cultured as described previously. 23 For proliferation and expression studies, HUVECs of second passage were used to avoid senescence.To induce growth arrest, HUVECs were kept in low serum medium (2% FCS) to allow cell survival for 48 hours before stimulation with bFGF (50 ng/mL) or VEGF-165 (50 ng/mL). HMEC-1 were kept in serum-free medium for 48 hours before stimulation with bFGF (50 ng/mL). At 5% to 10% confluence, photomicrographs of cells were taken in a fixed field before and 48 hours after stimulation and the percentage increase in cell count was calculated for each experiment. For blocker studies, cells were treated with TRAM-34 (0.1 nmol/L to 1 mol/L), 12 the inactive analogue TRAM-7 (1-tritylpyrrolidine; 1 mol/L), 12 CLT (0.1 nmol/L to 1 mol/L), or iberiotoxin (IbTx; 100 nmol/L). ReagentsPD98059, SB203580, and SP600125 were obtained from Tocris. CLT, IbTx, charydotoxin (ChTx), and apamin were obtained from Sigma. bFGF and VEGF-165 were obtained from Biochrom. All other chemicals were obtained from Sigma.